Icos binding proteins

ABSTRACT

The present invention relates to an ICOS binding protein or antigen binding portion thereof that is an agonist to human ICOS and does not induce complement, ADCC, or CDC when placed in contact with a T cell in vivo and methods of treating cancer, infectious disease and/or sepsis with said ICOS binding protein or antigen binding portion thereof. Further the ICOS binding proteins or antigen binding portions thereof of the present invention are capable of activating a T cell when placed in contact with said T cell; stimulating T cell proliferation when placed in contact with said T cell and/or inducing cytokine production when placed in contact with said T cell. The present invention relates to ICOS binding proteins or antigen binding portions thereof comprising one or more of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; and/or SEQ ID NO:6.

This application is a Divisional of U.S. application Ser. No. 15/006,560filed 26 Jan. 2016, which is a 111a application which claims benefit ofU.S. Provisional 62/247,355 filed 28 Oct. 2015, U.S. Provisional62/192,331 filed 14 Jul. 2015, and U.S. Provisional 62/108,605 filed 28Jan. 2015, which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to immunotherapy in thetreatment of human disease and reduction of adverse events relatedthereto. More specifically, the present invention relates to the use ofICOS binding proteins including ICOS agonist antibodies and their use asimmunomodulators in the treatment of cancer, infectious disease and/orsepsis.

BACKGROUND OF THE INVENTION

Enhancing anti-tumor T cell function and inducing T cell proliferationis a powerful and new approach for cancer treatment. Threeimmune-oncology antibodies (e.g., immuno-modulators) are presentlymarketed. Anti-CTLA-4 (YERVOY/ipilimumab) is thought to augment immuneresponses at the point of T cell priming and anti-PD-1 antibodies(OPDIVO/nivolumab and KEYTRUDA/pembrolizumab) are thought to act in thelocal tumor microenvironment, by relieving an inhibitory checkpoint intumor specific T cells that have already been primed and activated.

ICOS is a co-stimulatory T cell receptor with structural and functionalrelation to the CD28/CTLA-4-Ig superfamily (Hutloff, et al., “ICOS is aninducible T-cell co-stimulator structurally and functionally related toCD28”, Nature, 397: 263-266 (1999)). Activation of ICOS occurs throughbinding by ICOS-L (B7RP-1/B7-H2). Neither B7-1 nor B7-2 (ligands forCD28 and CTLA4) bind or activate ICOS. However, ICOS-L has been shown tobind weakly to both CD28 and CTLA-4 (Yao S et al., “B7-H2 is acostimulatory ligand for CD28 in human”, Immunity, 34(5); 729-40(2011)). Expression of ICOS appears to be restricted to T cells. ICOSexpression levels vary between different T cell subsets and on T cellactivation status. ICOS expression has been shown on resting TH17, Tfollicular helper (TFH) and regulatory T (Treg) cells; however, unlikeCD28; it is not highly expressed on naïve T_(H)1 and T_(H)2 effector Tcell populations (Paulos C M et al., “The inducible costimulator (ICOS)is critical for the development of human Th17 cells”, Sci Transl Med,2(55); 55ra78 (2010)). ICOS expression is highly induced on CD4+ andCD8+ effector T cells following activation through TCR engagement(Wakamatsu E, et al., “Convergent and divergent effects of costimulatorymolecules in conventional and regulatory CD4+ T cells”, Proc Natal AcadSci USA, 110(3); 1023-8 (2013)). Co-stimulatory signalling through ICOSreceptor only occurs in T cells receiving a concurrent TCR activationsignal (Sharpe A H and Freeman G J. “The B7-CD28 Superfamily”, Nat. RevImmunol, 2(2); 116-26 (2002)). In activated antigen specific T cells,ICOS regulates the production of both T_(H)1 and T_(H)2 cytokinesincluding IFN-γ, TNF-α, IL-10, IL-4, IL-13 and others. ICOS alsostimulates effector T cell proliferation, albeit to a lesser extent thanCD28 (Sharpe A H and Freeman G J. “The B7-CD28 Superfamily”, Nat. RevImmunol, 2(2); 116-26 (2002))

A growing body of literature supports the idea that activating ICOS onCD4+ and CD8+ effector T cells has anti-tumor potential. An ICOS-L-Fcfusion protein caused tumor growth delay and complete tumor eradicationin mice with SA-1 (sarcoma), Meth A (fibrosarcoma), EMT6 (breast) andP815 (mastocytoma) and EL-4 (plasmacytoma) syngeneic tumors, whereas noactivity was observed in the B16-F10 (melanoma) tumor model which isknown to be poorly immunogenic (Ara G et al., “Potent activity ofsoluble B7RP-1-Fc in therapy of murine tumors in syngeneic hosts”, Int.J Cancer, 103(4); 501-7 (2003)). The anti-tumor activity of ICOS-L-Fcwas dependent upon an intact immune response, as the activity wascompletely lost in tumors grown in nude mice. Analysis of tumors fromICOS-L-Fc treated mice demonstrated a significant increase in CD4+ andCD8+ T cell infiltration in tumors responsive to treatment, supportingthe immunostimulatory effect of ICOS-L-Fc in these models.

Another report using ICOS^(−/−) and ICOS-L^(−/−) mice demonstrated therequirement of ICOS signalling in mediating the anti-tumor activity ofan anti-CTLA4 antibody in the B16/B16 melanoma syngeneic tumor model (FuT et al., “The ICOS/ICOSL pathway is required for optimal antitumorresponses mediated by anti-CTLA-4 therapy”, Cancer Res, 71(16); 5445-54(2011)). Mice lacking ICOS or ICOS-L had significantly decreasedsurvival rates as compared to wild-type mice after anti-CTLA4 antibodytreatment. In a separate study, B16/B16 tumor cells were transduced tooverexpress recombinant murine ICOS-L. These tumors were found to besignificantly more sensitive to anti-CTLA4 treatment as compared to aB16/B16 tumor cells transduced with a control protein (Allison J et al.,“Combination immunotherapy for the treatment of cancer”, WO2011/041613A2 (2009)). These studies provide evidence of the anti-tumor potentialof an ICOS agonist, both alone and in combination with otherimmunomodulatory antibodies.

Emerging data from patients treated with anti-CTLA4 antibodies alsopoint to the positive role of ICOS+ effector T cells in mediating ananti-tumor immune response. Patients with metastatic melanoma (Giacomo AM D et al., “Long-term survival and immunological parameters inmetastatic melanoma patients who respond to ipilimumab 10 mg/kg withinan expanded access program”, Cancer Immunol Immunother., 62(6); 1021-8(2013)); urothelial (Carthon B C et al., “Preoperative CTLA-4 blockade:Tolerability and immune monitoring in the setting of a presurgicalclinical trial” Clin Cancer Res., 16(10); 2861-71 (2010)); breast(Vonderheide R H et al., “Tremelimumab in combination with exemestane inpatients with advanced breast cancer and treatment-associated modulationof inducible costimulator expression on patient T cells”, Clin CancerRes., 16(13); 3485-94 (2010)); and prostate cancer which have increasedabsolute counts of circulating and tumor infiltrating CD4⁺ICOS⁺ andCD8⁺ICOS⁺ T cells after ipilimumab treatment have significantly bettertreatment related outcomes than patients where little or no increasesare observed. Importantly, it was shown that ipilimumab changes theICOS⁺ T effector:T_(reg) ratio, reversing an abundance of T_(regs)pre-treatment to a significant abundance of T effectors vs. T_(regs)following treatment (Liakou C I et al., “CTLA-4 blockade increasesIFN-gamma producing CD4+ICOShi cells to shift the ratio of effector toregulatory T cells in cancer patients”, Proc Natl Acad Sci USA. 105(39);14987-92 (2008)) and (Vonderheide R H et al., Clin Cancer Res., 16(13);3485-94 (2010)). Therefore, ICOS positive T effector cells are apositive predictive biomarker of ipilimumab response which points to thepotential advantage of activating this population of cells with anagonist ICOS antibody.

Thus, there is a need for additional T cell proliferation inducingmolecules in the treatment of cancer.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, ICOS binding proteins orantigen binding portions thereof are provided comprising one or more of:CDRH1 as set forth in SEQ ID NO:1; CDRH2 as set forth in SEQ ID NO:2;CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4;CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ IDNO:6 or a direct equivalent of each CDR wherein a direct equivalent hasno more than two amino acid substitutions in said CDR.

In one embodiment of the present invention, ICOS binding proteins orantigen binding portions thereof are provided which specifically bindsto human ICOS wherein said ICOS binding protein comprises a V_(H) domaincomprising an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:7 and/or a V_(L) domain comprisingan amino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:8.

In one embodiment, humanized monoclonal antibodies or antigen bindingportions thereof are provided comprising heavy chain CDRs having theamino acid sequences set forth in SEQ ID NO:1; SEQ ID NO:2; and SEQ IDNO:3 and light chain CDRs having the amino acid sequences set forth inSEQ ID NO:4; SEQ ID NO:5; and SEQ ID NO:6. In one embodiment, humanizedmonoclonal antibodies are provided which comprise a hIgG4PE scaffold; aV_(H) domain comprising an amino acid sequence set forth in SEQ ID NO:7;and a V_(L) domain comprising an amino acid sequence set forth in SEQ IDNO:8. The antibodies of the present invention may stimulate cytokineproduction when contacted with a T cell.

In one embodiment, ICOS binding proteins are provided that compete forbinding to human ICOS with any one of the ICOS binding proteins orantigen binding portions thereof of the invention.

In one embodiment, methods are provided for treating cancer, infectiousdisease and/or sepsis with an ICOS binding protein or a pharmaceuticalcomposition comprising at least one ICOS binding protein of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: IFN-γ production from CD4+CD25− T cells.

FIG. 2: Proliferation in CD4+CD25− T cells.

FIG. 3: H2L5 humanized variant of anti-ICOS 422.2 shows better cytokineproduction in PBMC cells.

FIG. 4: 422 H2L5 IgG1 induced decreased T cell viability which was notapparent with Fc-disabled or hIgG4PE isotypes.

FIG. 5: Dose response of H2L5 hIgG4PE induced proinflammatory cytokineinduction in human CD4+ T cells.

FIG. 6: H2L5 hIgG4PE induces proliferation, cytokine production andincreased cytotoxic potential in activated PBMCs from healthy humandonors.

FIG. 7: Meso Scale Discovery (MSD) assay showing the inhibition ofICOS-L binding to ICOS by H2L5 hIgG4PE, indicating that it binds to thesame epitope on ICOS as ICOS-L and competes for binding.

FIG. 8: Recovered antibody V_(H) and V_(L) genes from RNA of hybridomaclone 422.2.

FIG. 9: Protein sequences of heavy and light chains of H2L5 hIgG4PE withsignal sequence.

FIG. 10: DNA sequence of coding region of H2L5 hIgG4PE heavy chain withsignal sequence.

FIG. 11: DNA sequence of coding region of H2L5 hIgG4PE light chain withsignal sequence.

FIG. 12: Plasma concentrations of H2L5 hIgG4PE in cynomolgus monkeys.Concentrations were determined after the (A) first or (B) second dose(day 15) of H2L5 hIgG4PE. Animals were sacrificed 48 hours post seconddose for tissue sample collection and histopathology analysis.

FIG. 13: Detection of H2L5 hIgG4PE binding to CD4+ T cells from thespleen and axillary lymph nodes of monkeys. Tissue was collected 48hours post-second dose (Day 17).

FIG. 14: Receptor Occupancy of H2L5 hIgG4PE in blood CD4+ T cells fromcynomolgus monkeys.

-   -   (A) ICOS “free receptor” as measured by positive binding of the        anti-ICOS fluorescently labelled antibody used for flow        cytometry, which binds only when H2L5 hIgG4PE is not present.    -   (B) Receptor bound H2L5 hIgG4PE on peripheral blood CD4+ cells        as measured by fluorescently labelled anti-Human IgG.

FIG. 15 (a) Phospho-AKT (T308) expression levels in Ba/F3-ICOS cellstreated with H2L5 hIgG4PE-Intracellular signalling antibody array (b)Phospho-AKT (S473) expression levels in Ba/F3-ICOS cells treated withH2L5 hIgG4PE-Intracellular signalling antibody array.

FIG. 16: H2L5 hIgG4PE in combination with ipilimumab results increasedproinflammatory cytokine production as compared to single antibodytreatment in PBMC pre-stimulation assay.

FIG. 17: H2L5 hIgG4PE in combination with pembrolizumab resultsincreased proinflammatory cytokine production as compared to singleantibody treatment in PBMC pre-stimulation assay.

FIG. 18: H2L5 hIgG4PE plus ipilimumab combination induces increasedproinflamatory cytokine production in a modified MLR assay with CEFTpeptide and pre-incubation.

FIG. 19: H2L5 hIgG4PE plus pembrolizumab combination induces increasedproinflamatory cytokine production in a modified MLR assay with CEFTpeptide and pre-incubation.

FIG. 20: H2L5 hIgG4PE anti-ICOS agonist mAb alone and in combinationwith pembrolizumab results in tumor growth inhibition in a human PBMCA2058 Melanoma mouse tumor model.

FIG. 21: anti-ICOS murine surrogate mAb results in significant tumorgrowth inhibition and increased survival in combination with an anti-PD1murine surrogate mAb in the CT26 mouse tumor model.

FIG. 22: anti-ICOS murine surrogate mAb results in significant tumorgrowth inhibition and increased survival in combination with an anti-PD1murine surrogate mAb in the EMT6 mouse tumor model.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein “ICOS” means any Inducible costimulator protein,Pseudonyms for ICOS (Inducible T-cell COStimulator) include AILIM;CD278; CVID1, JTT-1 or HT-2. MGC39850, or 8F4. ICOS is aCD28-superfamily costimulatory molecule that is expressed on activated Tcells. The protein encoded by this gene belongs to the CD28 and CTLA-4cell-surface receptor family. It forms homodimers and plays an importantrole in cell-cell signaling, immune responses, and regulation of cellproliferation. The amino acid sequence of human ICOS is shown below asSEQ ID NO:10.

(SEQ ID NO: 10) MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAF VVVCILGCILICWLTKKM

As used herein “ICOS-L” and “ICOS Ligand” are used interchangeably andrefer to the membrane bound natural ligand of human ICOS. ICOS ligand isa protein that in humans is encoded by the ICOSLG gene. ICOSLG has alsobeen designated as CD275 (cluster of differentiation 275). Pseudonymsfor ICOS-L include B7RP-1 and B7-H2.

As used herein the term “agonist” refers to an antigen binding protein,for example an ICOS binding protein, which upon contact with ICOS causesone or more of the following (1) stimulates or activates the ICOSreceptor, (2) enhances, increases or promotes, induces or prolongs anactivity, function or presence of ICOS and/or (3) enhances, increases,promotes or induces the expression of the ICOS. Agonist activity can bemeasured in vitro by various assays know in the art such as, but notlimited to, measurement of cell signaling, cell proliferation, immunecell activation markers, cytokine production. Agonist activity can alsobe measured in vivo by various assays that measure surrogate end pointssuch as, but not limited to the measurement of T cell proliferation orcytokine production.

As used herein the term “cross competes for binding” refers to any ICOSbinding protein that will compete for binding to ICOS with any of theICOS binding proteins of the present invention. Competition for bindingbetween two molecules for ICOS can be tested by various methods known inthe art including Flow cytometry, Meso Scale Discovery and ELISA.Binding can be measured directly, meaning two or more binding proteinscan be put in contact with ICOS and binding may be measured for one oreach. Alternatively, binding of molecules or interest can be testedagainst the binding or natural ligand and quantitatively compared witheach other.

The term “ICOS binding protein” as used herein refers to antibodies andother protein constructs, such as domains, which are capable of bindingto ICOS. In some instances, the ICOS is human ICOS. The term “ICOSbinding protein” can be used interchangeably with “ICOS antigen bindingprotein.” Thus, as is understood in the art, anti-ICOS antibodies and/orICOS antigen binding proteins would be considered ICOS binding proteins.As used herein, “antigen binding protein” is any protein, including butnot limited to antibodies, domains and other constructs describedherein, that binds to an antigen, such as ICOS. As used herein “antigenbinding portion” of an ICOS binding protein would include any portion ofthe ICOS binding protein capable of binding to ICOS, including but notlimited to, an antigen binding antibody fragment.

The term “antibody” is used herein in the broadest sense to refer tomolecules with an immunoglobulin-like domain (for example IgG, IgM, IgA,IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric,human, humanized, multispecific antibodies, including bispecificantibodies, and heteroconjugate antibodies; a single variable domain(e.g., V_(H), V_(HH), VL, domain antibody (dAb™)), antigen bindingantibody fragments, Fab, F(ab′)₂, Fv, disulphide linked Fv, single chainFv, disulphide-linked scFv, diabodies, TANDABS™, etc. and modifiedversions of any of the foregoing.

Alternative antibody formats include alternative scaffolds in which theone or more CDRs of the antigen binding protein can be arranged onto asuitable non-immunoglobulin protein scaffold or skeleton, such as anaffibody, a SpA scaffold, an LDL receptor class A domain, an avimer oran EGF domain.

The term “domain” refers to a folded protein structure which retains itstertiary structure independent of the rest of the protein. Generallydomains are responsible for discrete functional properties of proteinsand in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

The term “single variable domain” refers to a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains such as V_(H),V_(HH) and V_(L) and modified antibody variable domains, for example, inwhich one or more loops have been replaced by sequences which are notcharacteristic of antibody variable domains, or antibody variabledomains which have been truncated or comprise N- or C-terminalextensions, as well as folded fragments of variable domains which retainat least the binding activity and specificity of the full-length domain.A single variable domain is capable of binding an antigen or epitopeindependently of a different variable region or domain. A “domainantibody” or “dAb™” may be considered the same as a “single variabledomain”. A single variable domain may be a human single variable domain,but also includes single variable domains from other species such asrodent nurse shark and Camelid V_(HH) dAbs™. Camelid V_(HH) areimmunoglobulin single variable domain polypeptides that are derived fromspecies including camel, llama, alpaca, dromedary, and guanaco, whichproduce heavy chain antibodies naturally devoid of light chains. SuchV_(HH) domains may be humanized according to standard techniquesavailable in the art, and such domains are considered to be “singlevariable domains”. As used herein V_(H) includes camelid V_(HH) domains.

An antigen binding fragment may be provided by means of arrangement ofone or more CDRs on non-antibody protein scaffolds. “Protein Scaffold”as used herein includes but is not limited to an immunoglobulin (Ig)scaffold, for example an IgG scaffold, which may be a four chain or twochain antibody, or which may comprise only the Fc region of an antibody,or which may comprise one or more constant regions from an antibody,which constant regions may be of human or primate origin, or which maybe an artificial chimera of human and primate constant regions.

The protein scaffold may be an Ig scaffold, for example an IgG, or IgAscaffold. The IgG scaffold may comprise some or all the domains of anantibody (i.e. CH1, CH2, CH3, V_(H), V_(L)). The antigen binding proteinmay comprise an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4 orIgG4PE. For example, the scaffold may be IgG1. The scaffold may consistof, or comprise, the Fc region of an antibody, or is a part thereof.

The protein scaffold may be a derivative of a scaffold selected from thegroup consisting of CTLA-4, lipocalin, Protein A derived molecules suchas Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody);heat shock proteins such as GroEl and GroES; transferrin (trans-body);ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain(Tetranectin); human γ-crystallin and human ubiquitin (affilins); PDZdomains; scorpion toxin kunitz type domains of human proteaseinhibitors; and fibronectin/adnectin; which has been subjected toprotein engineering in order to obtain binding to an antigen, such asICOS, other than the natural ligand.

Antigen binding site refers to a site on an antigen binding proteinwhich is capable of specifically binding to an antigen, this may be asingle variable domain, or it may be paired V_(H)/V_(L) domains as canbe found on a standard antibody. Single-chain Fv (ScFv) domains can alsoprovide antigen-binding sites. The term “epitope-binding domain” refersto a domain that specifically binds to a region of an antigen known asthe epitope independently of a different domain.

The term multi-specific antigen binding protein refers to antigenbinding proteins which comprise at least two different antigen bindingsites. Each of these antigen-binding sites will be capable of binding toa different epitope, which may be present on the same antigen ordifferent antigens. The multi-specific antigen binding protein will havespecificity for more than one antigen, for example two antigens, or forthree antigens, or for four antigens.

Examples of multi-specific antigen binding proteins include those thatconsist of, or consist essentially of, an Fc region of an antibody, or apart thereof, linked at each end, directly or indirectly (for example,via a linker sequence) to a binding domain. Such an antigen bindingprotein may comprise two binding domains separated by an Fc region, orpart thereof. By separated is meant that the binding domains are notdirectly linked to one another, and may be located at opposite ends (Cand N terminus) of an Fc region, or any other scaffold region.

The antigen binding protein may comprise two scaffold regions each boundto two binding domains, for example at the N and C termini of eachscaffold region, either directly or indirectly via a linker. Eachbinding domain may bind to a different antigen.

As used herein, the term mAbdAb refers to a monoclonal antibody linkedto a further binding domain, in particular a single variable domain suchas a domain antibody. A mAbdAb has at least two antigen binding sites,at least one of which is from a domain antibody, and at least one isfrom a paired V_(H)/VL domain.

A “dAb™ conjugate” refers to a composition comprising a dAb to which adrug is chemically conjugated by means of a covalent or noncovalentlinkage. Preferably, the dAb and the drug are covalently bonded. Suchcovalent linkage could be through a peptide bond or other means such asvia a modified side chain. The noncovalent bonding may be direct (e.g.,electrostatic interaction, hydrophobic interaction) or indirect (e.g.,through noncovalent binding of complementary binding partners (e.g.,biotin and avidin), wherein one partner is covalently bonded to drug andthe complementary binding partner is covalently bonded to the dAb™).When complementary binding partners are employed, one of the bindingpartners can be covalently bonded to the drug directly or through asuitable linker moiety, and the complementary binding partner can becovalently bonded to the dAb directly or through a suitable linkermoiety.

As used herein, “dAb™ fusion” refers to a fusion protein that comprisesa dAb™ and a polypeptide drug (which could be a dAb™ or mAb). The dAb™and the polypeptide drug are present as discrete parts (moieties) of asingle continuous polypeptide chain.

In one embodiment, antigen binding proteins of the present disclosureshow cross-reactivity between human ICOS and ICOS from another species,such as cynomolgus ICOS. In an embodiment, the antigen binding proteinsof the invention specifically bind human and cynomolgus ICOS. Theprovision of a drug that can bind human and monkey species allows one totest results in these system and make side-by-side comparisons of datausing the same drug. Cross reactivity between other species used indisease models such as dog or monkey, in particular monkey, isenvisaged.

Competition between an ICOS binding protein and a reference ICOS bindingprotein may be determined by competition MSD, ELISA, FMAT or BIAcore. Inone embodiment, the competition assay is carried out by comparison of anICOS binding protein with ICOS ligand binding. There are severalpossible reasons for this competition: the two proteins may bind to thesame or overlapping epitopes, there may be steric inhibition of binding,or binding of the first protein may induce a conformational change inthe antigen that prevents or reduces binding of the second protein.

The term “neutralizes” as used throughout the present specificationmeans that the interaction between ICOS and ICOS-L is reduced in thepresence of an antigen binding protein as described herein in comparisonto the interaction of ICOS and ICOS-L in the absence of the ICOS bindingprotein, in vitro or in vivo. Neutralization may be due to one or moreof blocking ICOS binding to its ligand, preventing ICOS from beingactivated by its ligand, down regulating ICOS or its receptor, oraffecting effector functionality. For example, the ligand bindingcompetition described in Examples 3 and 5 may be used to assess theneutralizing capability of an ICOS binding protein.

The effect of an ICOS binding protein on the interaction between ICOSand ICOS-L may be partial or total. A neutralising ICOS binding proteinmay block the interaction of ICOS with ICOS-L by at least 20%, 30% 40%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%,95%, 96%, 97%, 98%, 99% or 100% relative to ICOS-ICOS-L interactions inthe absence of the ICOS binding protein.

Neutralization may be determined or measured using one or more assaysknown to the skilled person or as described herein.

Affinity is the strength of binding of one molecule, e.g. an antigenbinding protein of the invention, to another, e.g. its target antigen,at a single binding site. The binding affinity of an antigen bindingprotein to its target may be determined by equilibrium methods (e.g.enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)),or kinetics (e.g. BIACORE™ analysis). For example, the Biacore™ methodsdescribed in Example 5 may be used to measure binding affinity.

Avidity is the sum total of the strength of binding of two molecules toone another at multiple sites, e.g. taking into account the valency ofthe interaction.

In an embodiment, the equilibrium dissociation constant (KD) of the ICOSbinding protein-ICOS interaction is 100 nM or less, 10 nM or less, 2 nMor less or 1 nM or less. Alternatively the KD may be between 5 and 10nM; or between 1 and 2 nM. The KD may be between 1 pM and 500 pM; orbetween 500 pM and 1 nM. A skilled person will appreciate that thesmaller the KD numerical value, the stronger the binding. The reciprocalof KD (i.e. 1/KD) is the equilibrium association constant (KA) havingunits M⁻¹. A skilled person will appreciate that the larger the KAnumerical value, the stronger the binding.

The dissociation rate constant (kd) or “off-rate” describes thestability of the ICOS binding protein ICOS complex, i.e. the fraction ofcomplexes that decay per second. For example, a kd of 0.01 s⁻¹ equatesto 1% of the complexes decaying per second. In an embodiment, thedissociation rate constant (kd) is 1×10⁻³ s⁻¹ or less, 1×10⁻⁴ s⁻¹ orless, 1×10⁻⁵ s⁻¹ or less, or 1×10⁻⁶ s⁻¹ or less. The kd may be between1×10⁻⁵s⁻¹ and 1×10⁻⁴ s⁻¹; or between 1×10⁻⁴ s⁻¹ and 1×10⁻³ s⁻¹.

The association rate constant (ka) or “on-rate” describes the rate ofICOS binding protein-ICOS complex formation. In an embodiment, theassociation rate constant (ka) is about 1.0×10⁵M⁻¹s⁻¹.

By “isolated” it is intended that the molecule, such as an antigenbinding protein or nucleic acid, is removed from the environment inwhich it may be found in nature. For example, the molecule may bepurified away from substances with which it would normally exist innature. For example, the mass of the molecule in a sample may be 95% ofthe total mass.

The term “expression vector” as used herein means an isolated nucleicacid which can be used to introduce a nucleic acid of interest into acell, such as a eukaryotic cell or prokaryotic cell, or a cell freeexpression system where the nucleic acid sequence of interest isexpressed as a peptide chain such as a protein. Such expression vectorsmay be, for example, cosmids, plasmids, viral sequences, transposons,and linear nucleic acids comprising a nucleic acid of interest. Once theexpression vector is introduced into a cell or cell free expressionsystem (e.g., reticulocyte lysate) the protein encoded by the nucleicacid of interest is produced by the transcription/translation machinery.Expression vectors within the scope of the disclosure may providenecessary elements for eukaryotic or prokaryotic expression and includeviral promoter driven vectors, such as CMV promoter driven vectors,e.g., pcDNA3.1, pCEP4, and their derivatives, Baculovirus expressionvectors, Drosophila expression vectors, and expression vectors that aredriven by mammalian gene promoters, such as human Ig gene promoters.Other examples include prokaryotic expression vectors, such as T7promoter driven vectors, e.g., pET41, lactose promoter driven vectorsand arabinose gene promoter driven vectors. Those of ordinary skill inthe art will recognize many other suitable expression vectors andexpression systems.

The term “recombinant host cell” as used herein means a cell thatcomprises a nucleic acid sequence of interest that was isolated prior toits introduction into the cell. For example, the nucleic acid sequenceof interest may be in an expression vector while the cell may beprokaryotic or eukaryotic. Exemplary eukaryotic cells are mammaliancells, such as but not limited to, COS-1, COS-7, HEK293, BHK21, CHO,BSC-1, HepG2, 653, SP2/0, NS0, 293, HeLa, myeloma, lymphoma cells or anyderivative thereof. Most preferably, the eukaryotic cell is a HEK293,NS0, SP2/0, or CHO cell. E. coli is an exemplary prokaryotic cell. Arecombinant cell according to the disclosure may be generated bytransfection, cell fusion, immortalization, or other procedures wellknown in the art. A nucleic acid sequence of interest, such as anexpression vector, transfected into a cell may be extrachromasomal orstably integrated into the chromosome of the cell.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one ormore human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., Queen et al.Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson, et al.,Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may beone selected from a conventional database, e.g., the KABAT™ database,Los Alamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanized antibodies—see, for example, EP-A-0239400 andEP-A-054951.

The term “fully human antibody” includes antibodies having variable andconstant regions (if present) derived from human germline immunoglobulinsequences. The human sequence antibodies of the invention may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). Fully humanantibodies comprise amino acid sequences encoded only by polynucleotidesthat are ultimately of human origin or amino acid sequences that areidentical to such sequences. As meant herein, antibodies encoded byhuman immunoglobulin-encoding DNA inserted into a mouse genome producedin a transgenic mouse are fully human antibodies since they are encodedby DNA that is ultimately of human origin. In this situation, humanimmunoglobulin-encoding DNA can be rearranged (to encode an antibody)within the mouse, and somatic mutations may also occur. Antibodiesencoded by originally human DNA that has undergone such changes in amouse are fully human antibodies as meant herein. The use of suchtransgenic mice makes it possible to select fully human antibodiesagainst a human antigen. As is understood in the art, fully humanantibodies can be made using phage display technology wherein a humanDNA library is inserted in phage for generation of antibodies comprisinghuman germline DNA sequence.

The term “donor antibody” refers to an antibody that contributes theamino acid sequences of its variable regions, CDRs, or other functionalfragments or analogs thereof to a first immunoglobulin partner. Thedonor, therefore, provides the altered immunoglobulin coding region andresulting expressed altered antibody with the antigenic specificity andneutralising activity characteristic of the donor antibody.

The term “acceptor antibody” refers to an antibody that is heterologousto the donor antibody, which contributes all (or any portion) of theamino acid sequences encoding its heavy and/or light chain frameworkregions and/or its heavy and/or light chain constant regions to thefirst immunoglobulin partner. A human antibody may be the acceptorantibody.

The terms “V_(H)” and “V_(L)” are used herein to refer to the heavychain variable region and light chain variable region respectively of anantigen binding protein.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antigen binding protein. These are the hypervariableregions of immunoglobulin heavy and light chains. There are three heavychain and three light chain CDRs (or CDR regions) in the variableportion of an immunoglobulin. Thus, “CDRs” as used herein refers to allthree heavy chain CDRs, all three light chain CDRs, all heavy and lightchain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domainsequences and full length antibody sequences are numbered according tothe Kabat numbering convention. Similarly, the terms “CDR”, “CDRL1”,“CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, “CDRH3” used in the Examples followthe Kabat numbering convention. For further information, see Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1991).

It will be apparent to those skilled in the art that there arealternative numbering conventions for amino acid residues in variabledomain sequences and full length antibody sequences. There are alsoalternative numbering conventions for CDR sequences, for example thoseset out in Chothia et al. (1989) Nature 342: 877-883. The structure andprotein folding of the antibody may mean that other residues areconsidered part of the CDR sequence and would be understood to be so bya skilled person.

Other numbering conventions for CDR sequences available to a skilledperson include “AbM” (University of Bath) and “contact” (UniversityCollege London) methods. The minimum overlapping region using at leasttwo of the Kabat, Chothia, AbM and contact methods can be determined toprovide the “minimum binding unit”. The minimum binding unit may be asub-portion of a CDR.

Table 1 below represents one definition using each numbering conventionfor each CDR or binding unit. The Kabat numbering scheme is used inTable 1 to number the variable domain amino acid sequence. It should benoted that some of the CDR definitions may vary depending on theindividual publication used.

TABLE 1 Chothia Contact Minimum Kabat CDR CDR AbM CDR CDR binding unitH1 31-35/35A/ 26-32/33/34 26-35/ 30-35/ 31-32 35B 35A/35B 35A/35B H250-65 52-56 50-58 47-58 52-56 H3  95-102  95-102  95-102  93-101  95-101L1 24-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L389-97 89-97 89-97 89-96 89-96

Accordingly, ICOS binding proteins are provided, which comprises any oneor a combination of the following CDRs:

CDRH1: (SEQ ID NO: 1) DYAMH CDRH2: (SEQ ID NO: 2) LISIYSDHTNYNQKFQGCDRH3: (SEQ ID NO: 3) NNYGNYGWYFDV CDRL1: (SEQ ID NO: 4) SASSSVSYMH CDRL2: (SEQ ID NO: 5) DTSKLAS  CDRL3: (SEQ ID NO: 6) FQGSGYPYT

In one embodiment of the present invention the ICOS binding proteincomprises CDRH1 (SEQ ID NO:1), CDRH2 (SEQ ID NO:2), and CDRH3 (SEQ IDNO:3) in the heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO:7. ICOS binding proteins of the present inventioncomprising the humanized heavy chain variable region set forth in SEQ IDNO:7 are designated as “H2.” In some embodiments, the ICOS bindingproteins of the present invention comprise a heavy chain variable regionhaving at least 90% sequence identity to SEQ ID NO:7. Suitably, the ICOSbinding proteins of the present invention may comprise a heavy chainvariable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO:7.

Humanized Heavy Chain (V_(H)) Variable Region (H2): (SEQ ID NO: 7)QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYAMHWVRQA PGQGLEWMGL ISIYSDHTNY NQKFQGRVTI TADKSTSTAY MELSSLRSED TAVYYCGRNN YGNYGWYFDV WGQGTTVTVS S

In one embodiment of the present invention the ICOS binding proteincomprises CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), and CDRL3 (SEQ IDNO:6) in the light chain variable region having the amino acid sequenceset forth in SEQ ID NO:8. ICOS binding proteins of the present inventioncomprising the humanized light chain variable region set forth in SEQ IDNO:8 are designated as “L5.” Thus, an ICOS binding protein of thepresent invention comprising the heavy chain variable region of SEQ IDNO:7 and the light chain variable region of SEQ ID NO:8 can bedesignated as H2L5 herein.

Suitably a leader sequence for the variable heavy chain and light chainconstructs is show in FIG. 9 and includes, but is not limited to:MGWSCIILFLVATATGVHS (SEQ ID NO:11)

In some embodiments, the ICOS binding proteins of the present inventioncomprise a light chain variable region having at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:8. Suitably,the ICOS binding proteins of the present invention may comprise a lightchain variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQID NO:8.

Humanized Light Chain (V_(L)) Variable Region (L5) (SEQ ID NO: 8)EIVLTQSPAT LSLSPGERAT LSCSASSSVS YMHWYQQKPG QAPRLLIYDT SKLASGIPAR FSGSGSGTDY TLTISSLEPE DFAVYYCFQG SGYPYTFGQG TKLEIK

CDRs or minimum binding units may be modified by at least one amino acidsubstitution, deletion or addition, wherein the variant antigen bindingprotein substantially retains the biological characteristics of theunmodified protein, such as a murine antibody produced from clone 422.2or an antibody comprising SEQ ID NO:7 and SEQ ID NO:8.

It will be appreciated that each of CDR H1, H2, H3, L1, L2, L3 may bemodified alone or in combination with any other CDR, in any permutationor combination. In one embodiment, a CDR is modified by thesubstitution, deletion or addition of up to 3 amino acids, for example 1or 2 amino acids, for example 1 amino acid. Typically, the modificationis a substitution, particularly a conservative substitution, for exampleas shown in Table 2 below.

TABLE 2 Side chain Members Hydrophobic Met, Ala, Val, Leu, Ile Neutralhydrophilic Cys, Ser, Thr Acidic Asp, Glu Basic Asn, Gln, His, Lys, ArgResidues that influence chain orientation Gly, Pro Aromatic Trp, Tyr,Phe

For example, in a variant CDR, the amino acid residues of the minimumbinding unit may remain the same, but the flanking residues thatcomprise the CDR as part of the Kabat or Chothia definition(s) may besubstituted with a conservative amino acid residue.

Such antigen binding proteins comprising modified CDRs or minimumbinding units as described above may be referred to herein as“functional CDR variants” or “functional binding unit variants”.Suitably, in one embodiment ICOS binding proteins are providedcomprising one or more CDRs having the amino acid sequences set forth inSEQ ID NOs:1, 2, 3, 4, 5, and/or 6 and/or a function CDR variantthereof.

The term “epitope” as used herein refers to that portion of the antigenthat makes contact with a particular binding domain of the antigenbinding protein. An epitope may be linear orconformational/discontinuous. A conformational or discontinuous epitopecomprises amino acid residues that are separated by other sequences,i.e. not in a continuous sequence in the antigen's primary sequence.Although the residues may be from different regions of the peptidechain, they are in close proximity in the three dimensional structure ofthe antigen. In the case of multimeric antigens, a conformational ordiscontinuous epitope may include residues from different peptidechains. Particular residues comprised within an epitope can bedetermined through computer modelling programs or via three-dimensionalstructures obtained through methods known in the art, such as X-raycrystallography.

The CDRs L1, L2, L3, H1 and H2 tend to structurally exhibit one of afinite number of main chain conformations. The particular canonicalstructure class of a CDR is defined by both the length of the CDR and bythe loop packing, determined by residues located at key positions inboth the CDRs and the framework regions (structurally determiningresidues or SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815)have generated an automatic method to define the “key residue” canonicaltemplates. Cluster analysis is used to define the canonical classes forsets of CDRs, and canonical templates are then identified by analysingburied hydrophobics, hydrogen-bonding residues, and conserved glycinesand prolines. The CDRs of antibody sequences can be assigned tocanonical classes by comparing the sequences to the key residuetemplates and scoring each template using identity or similaritymatrices.

There may be multiple variant CDR canonical positions per CDR, percorresponding CDR, per binding unit, per heavy or light chain variableregion, per heavy or light chain, and per antigen binding protein, andtherefore any combination of substitution may be present in the antigenbinding protein of the invention, provided that the canonical structureof the CDR is maintained such that the antigen binding protein iscapable of specifically binding ICOS.

As discussed above, the particular canonical structure class of a CDR isdefined by both the length of the CDR and by the loop packing,determined by residues located at key positions in both the CDRs and theframework regions.

“Percent identity” between a query nucleic acid sequence and a subjectnucleic acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTN algorithm when a subjectnucleic acid sequence has 100% query coverage with a query nucleic acidsequence after a pair-wise BLASTN alignment is performed. Such pair-wiseBLASTN alignments between a query nucleic acid sequence and a subjectnucleic acid sequence are performed by using the default settings of theBLASTN algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query nucleic acid sequence may be described by anucleic acid sequence identified in one or more claims herein.

“Percent identity” between a query amino acid sequence and a subjectamino acid sequence is the “Identities” value, expressed as apercentage, that is calculated by the BLASTP algorithm when a subjectamino acid sequence has 100% query coverage with a query amino acidsequence after a pair-wise BLASTP alignment is performed. Such pair-wiseBLASTP alignments between a query amino acid sequence and a subjectamino acid sequence are performed by using the default settings of theBLASTP algorithm available on the National Center for BiotechnologyInstitute's website with the filter for low complexity regions turnedoff. Importantly, a query amino acid sequence may be described by anamino acid sequence identified in one or more claims herein.

The query sequence may be 100% identical to the subject sequence, or itmay include up to a certain integer number of amino acid or nucleotidealterations as compared to the subject sequence such that the % identityis less than 100%. For example, the query sequence is at least 50, 60,70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subjectsequence. Such alterations include at least one amino acid deletion,substitution (including conservative and non-conservative substitution),or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the query sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids or nucleotides in the query sequence or in one or morecontiguous groups within the query sequence.

The % identity may be determined across the entire length of the querysequence, including the CDR(s). Alternatively, the % identity mayexclude the CDR(s), for example the CDR(s) is 100% identical to thesubject sequence and the % identity variation is in the remainingportion of the query sequence, so that the CDR sequence is fixed/intact.

The variant sequence substantially retains the biologicalcharacteristics of the unmodified protein, such as SEQ ID NO:7 or SEQ IDNO:8.

The V_(H) or V_(L) sequence may be a variant sequence with up to 15amino acid substitutions, additions or deletions. For example, thevariant sequence may have up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s).

The sequence variation may exclude the CDR(s), for example the CDR(s) isthe same as the V_(H) or V_(L) (or HC or LC) sequence and the variationis in the remaining portion of the V_(H) or V_(L) (or HC or LC)sequence, so that the CDR sequence is fixed/intact.

The skilled person will appreciate that, upon production of an antigenbinding protein such as an antibody, in particular depending on the cellline used and particular amino acid sequence of the antigen bindingprotein, post-translational modifications may occur. For example, thismay include the cleavage of certain leader sequences, the addition ofvarious sugar moieties in various glycosylation and phosphorylationpatterns, deamidation, oxidation, disulfide bond scrambling,isomerisation, C-terminal lysine clipping, and N-terminal glutaminecyclisation. The present invention encompasses the use of antigenbinding proteins which have been subjected to, or have undergone, one ormore post-translational modifications. Thus an “antigen binding protein”or “antibody” of the invention includes an “antigen binding protein” or“antibody”, respectively, as defined earlier which has undergone apost-translational modification such as described herein.

Deamidation is an enzymatic reaction primarily converting asparagine (N)to isoaspartic acid and aspartic acid (D) at approximately 3:1 ratio. Toa much lesser degree, deamidation can occur with glutamine residues in asimilar manner. Deamidation in a CDR results in a change in charge ofthe molecule, but typically does not result in a change in antigenbinding, nor does it impact on PK/PD.

Oxidation can occur during production and storage (i.e. in the presenceof oxidizing conditions) and results in a covalent modification of aprotein, induced either directly by reactive oxygen species orindirectly by reaction with secondary by-products of oxidative stress.Oxidation happens primarily with methionine residues, but occasionallycan occur at tryptophan and free cysteine residues.

Disulfide bond scrambling can occur during production and basic storageconditions. Under certain circumstances, disulfide bonds can break orform incorrectly, resulting in unpaired cysteine residues (—SH). Thesefree (unpaired) sulfhydryls (—SH) can promote shuffling.

Isomerization typically occurs during production, purification, andstorage (at acidic pH) and usually occurs when aspartic acid isconverted to isoaspartic acid through a chemical process.

N-terminal glutamine in the heavy chain and/or light chain is likely toform pyroglutamate (pGlu). Most pGlu formation happens in the productionbioreactor, but it can be formed non-enzymatically, depending on pH andtemperature of processing and storage conditions. pGlu formation isconsidered as one of the principal degradation pathways for recombinantmAb s.

C-terminal lysine clipping is an enzymatic reaction catalyzed bycarboxypeptidases, and is commonly observed in recombinant mAbs.Variants of this process include removal of lysine from one or bothheavy chains. Lysine clipping does not appear to impact bioactivity andhas no effect on mAb effector function.

Naturally occurring autoantibodies exist in humans that can bind toproteins. Autoantibodies can thus bind to endogenous proteins (presentin naïve subjects) as well as to proteins or peptides which areadministered to a subject for treatment. Therapeutic protein-bindingautoantibodies and antibodies that are newly formed in response to drugtreatment are collectively termed anti-drug antibodies (ADAs).Pre-existing antibodies against molecules such as therapeutic proteinsand peptides, administered to a subject can affect their efficacy andcould result in administration reactions, hypersensitivity, alteredclinical response in treated patients and altered bioavailability bysustaining, eliminating or neutralizing the molecule. It could beadvantageous to provide molecules for therapy which comprise humanimmunoglobulin (antibody) single variable domains or dAbs™ which havereduced immunogenicity (i.e. reduced ability to bind to pre-existingADAs when administered to a subject, in particular a human subject.

Thus, in one embodiment of the present invention there is provided amodified dAb™ which has reduced ability to bind to pre-existingantibodies (ADAs) as compared to the equivalent unmodified molecule. Byreduced ability to bind it is meant that the modified molecule bindswith a reduced affinity or reduced avidity to a pre-existing ADA. Saidmodified dAb™ comprise one or more modifications selected from: (a) aC-terminal addition, extension, deletion or tag, and/or (b) one or moreamino acid framework substitutions.

Polypeptides and dAbs™ of the disclosure and agonists comprising thesecan be formatted to have a larger hydrodynamic size, for example, byattachment of a PEG group, serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. Forexample, polypeptides dAbs™ and agonists may be formatted as a largerantigen-binding fragment of an antibody or as an antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv).

As used herein, “hydrodynamic size” refers to the apparent size of amolecule (e.g., an antigen binding protein) based on the diffusion ofthe molecule through an aqueous solution. The diffusion or motion of aprotein through solution can be processed to derive an apparent size ofthe protein, where the size is given by the “Stokes radius” or“hydrodynamic radius” of the protein particle. The “hydrodynamic size”of a protein depends on both mass and shape (conformation), such thattwo proteins having the same molecular mass may have differinghydrodynamic sizes based on the overall conformation and charge of theprotein. An increase in hydrodynamic size can give an associateddecrease in renal clearance leading to an observed increase in half life(t_(1/2)).

Hydrodynamic size of the antigen binding proteins (e.g., domain antibodymonomers and multimers) of the disclosure may be determined usingmethods which are well known in the art. For example, gel filtrationchromatography may be used to determine the hydrodynamic size of anantigen binding protein. Suitable gel filtration matrices fordetermining the hydrodynamic sizes of antigen binding proteins, such ascross-linked agarose matrices, are well known and readily available.

The size of an antigen binding protein format (e.g., the size of a PEGmoiety attached to a domain antibody monomer), can be varied dependingon the desired application. For example, where antigen binding proteinis intended to leave the circulation and enter into peripheral tissues,it is desirable to keep the hydrodynamic size of the ICOS bindingprotein low to facilitate extravazation from the blood stream.Alternatively, where it is desired to have the antigen binding proteinremain in the systemic circulation for a longer period of time the sizeof the antigen binding protein can be increased, for example byformatting as an Ig like protein.

Pharmaceutical Compositions

Antigen binding protein as described herein may be incorporated intopharmaceutical compositions for use in the treatment of the humandiseases described herein. In one embodiment, the pharmaceuticalcomposition comprises an antigen binding protein optionally incombination with one or more pharmaceutically acceptable carriers and/orexcipients.

Such compositions comprise a pharmaceutically acceptable carrier asknown and called for by acceptable pharmaceutical practice.

Pharmaceutical compositions may be administered by injection orcontinuous infusion (examples include, but are not limited to,intravenous, intraperitoneal, intradermal, subcutaneous, intramuscularand intraportal). In one embodiment, the composition is suitable forintravenous administration. Pharmaceutical compositions may be suitablefor topical administration (which includes, but is not limited to,epicutaneous, inhaled, intranasal or ocular administration) or enteraladministration (which includes, but is not limited to, oral or rectaladministration).

Pharmaceutical compositions may comprise between 0.5 mg to 10 g of ICOSbinding protein, for example between 5 mg and 1 g of antigen bindingprotein. Alternatively, the composition may comprise between 5 mg and500 mg, for example between 5 mg and 50 mg. Methods for the preparationof such pharmaceutical compositions are well known to those skilled inthe art. Other excipients may be added to the composition as appropriatefor the mode of administration and the particular protein used. Examplesof different excipients and their uses are described in Lowe et al.,(2011).

Effective doses and treatment regimes for administering the antigenbinding protein may be dependent on factors such as the age, weight andhealth status of the patient and disease to be treated. Such factors arewithin the purview of the attending physician. Guidance in selectingappropriate doses may be found in e.g Bai et al., (2012).

The pharmaceutical composition may comprise a kit of parts of theantigen binding protein together with other medicaments, optionally withinstructions for use. For convenience, the kit may comprise the reagentsin predetermined amounts with instructions for use.

The terms “individual”, “subject” and “patient” are used hereininterchangeably. In one embodiment, the subject is a mammal, such as aprimate, for example a marmoset or monkey. In another embodiment, thesubject is a human.

The antigen binding protein described herein may also be used in methodsof treatment. Treatment can be therapeutic, prophylactic orpreventative. Treatment encompasses alleviation, reduction, orprevention of at least one aspect or symptom of a disease andencompasses prevention or cure of the diseases described herein.

The ICOS binding protein or antigen binding portion thereof describedherein is used in an effective amount for therapeutic, prophylactic orpreventative treatment. A therapeutically effective amount of the ICOSbinding protein or antigen binding portion thereof described herein isan amount effective to ameliorate or reduce one or more symptoms of, orto prevent or cure, the disease.

Thus, in one embodiment ICOS binding proteins or antigen bindingportions thereof of the present invention are provided for use intherapy. In one embodiment, ICOS binding proteins or antigen bindingportions thereof of the present invention are provided for use in thetreatment of cancer, infectious disease and/or sepsis. The presentinvention also provides the use of an ICOS binding protein or antigenbinding portion thereof of the present invention in the manufacture of amedicament for the treatment of cancer, infectious disease and/orsepsis.

Thus, provided herein are isolated ICOS binding proteins or antigenbinding portions thereof or the pharmaceutical compositions comprisingsaid isolated ICOS binding proteins or antigen binding portions thereoffor use in the treatment of cancer, infectious disease and/or sepsis.

Production Methods

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, antigen binding proteins maypurified from cells that naturally express them (e.g., an antibody canbe purified from a hybridoma that produces it), or produced inrecombinant expression systems.

A number of different expression systems and purification regimes can beused to generate the antigen binding protein of the invention.Generally, host cells are transformed with a recombinant expressionvector encoding the desired antigen binding protein. A wide range ofhost cells can be employed, including Prokaryotes (including Gramnegative or Gram positive bacteria, for example Escherichia coli,Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes includingyeast (for example Saccharomyces cerevisiae, Pichia pastoris), fungi(for example Aspergillus sp.), or higher Eukaryotes including insectcells and cell lines of mammalian origin (for example, CHO, Perc6,HEK293, HeLa).

The host cell may be an isolated host cell. The host cell is usually notpart of a multicellular organism (e.g., plant or animal). The host cellmay be a non-human host cell.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts and methods of cloning areknown in the art.

The cells can be cultured under conditions that promote expression ofthe antigen binding protein, and the polypeptide recovered byconventional protein purification procedures. The antigen bindingproteins contemplated for use herein include substantially homogeneousantigen binding proteins substantially free of contaminating materials.

The skilled person will appreciate that, upon production of the antigenbinding protein, in particular depending on the cell line used andparticular amino acid sequence of the antigen binding protein,post-translational modifications may occur. This may include thecleavage of certain leader sequences, the addition of various sugarmoieties in various glycosylation patterns, deamidation (for example atan asparagine or glutamine residue), oxidation (for example at amethionine, tryptophan or free cysteine residue), disulfide bondscrambling, isomerisation (for example at an aspartic acid residue),C-terminal lysine clipping (for example from one or both heavy chains),and N-terminal glutamine cyclisation (for example in the heavy and/orlight chain). The present invention encompasses the use of antibodieswhich have been subjected to, or have undergone, one or morepost-translational modifications. The modification may occur in a CDR,the variable framework region, or the constant region. The modificationmay result in a change in charge of the molecule. The modificationtypically does not result in a change in antigen binding, function,bioactivity, nor does it impact the pharmacokinetic (PK) orpharmacodynamic (PD) characteristics of the ICOS binding protein.

The term “Effector Function” as used herein is meant to refer to one ormore of Antibody dependant cell mediated cytotoxic activity (ADCC),Complement-dependant cytotoxic activity (CDC) mediated responses,Fc-mediated phagocytosis or antibody dependant cellular phagocytosis(ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antigen bindingprotein and various Fc receptors (FcR) including FcγRI (CD64), FcγRII(CD32) and FcγRIII (CD16) is believed to mediate the effector functionsof the antigen binding protein. Significant biological effects can be aconsequence of effector functionality. Usually, the ability to mediateeffector function requires binding of the antigen binding protein to anantigen and not all antigen binding proteins will mediate every effectorfunction.

Effector function can be measured in a number of ways including forexample via binding of the FcγRIII on Natural Killer cells or via FcγRIon monocytes/macrophages to measure for ADCC/ADCP effector function. Forexample an antigen binding protein of the present invention can beassessed for ADCC effector function in a Natural Killer cell assay.Practical approaches to evaluate ADCC and/or CDC function can be foundin (Kellner C et al., “Boosting ADCC and CDC activity by Fc engineeringand evaluation of antibody effector functions”, Methods, 1; 65(1):105-13(2014))

Some isotypes of human constant regions, in particular IgG4 and IgG2isotypes, have reduced function of a) activation of complement by theclassical pathway; and b) antibody-dependent cellular cytotoxicity.Various modifications to the heavy chain constant region of antigenbinding proteins may be carried out depending on the desired effectorproperty. IgG1 constant regions containing specific mutations haveseparately been described to reduce binding to Fc receptors andtherefore reduce ADCC and CDC. (Kellner C et al., “Boosting ADCC and CDCactivity by Fc engineering and evaluation of antibody effectorfunctions”, Methods, 1; 65(1):105-13 (2014))

In one embodiment of the present invention there is provided an antigenbinding protein comprising a constant region such that the antigenbinding protein has reduced ADCC and/or complement activation oreffector functionality. In one such embodiment the heavy chain constantregion may comprise a naturally disabled constant region of IgG2 or IgG4isotype or a mutated IgG1 constant region. One example comprises thesubstitutions of alanine residues at positions 235 and 237 (EU indexnumbering).

The subclass of an antibody in part determines secondary effectorfunctions, such as complement activation or Fc receptor (FcR) bindingand antibody dependent cell cytotoxicity (ADCC) (Huber, et al., Nature229(5284): 419-20 (1971); Brunhouse, et al., Mol Immunol 16(11): 907-17(1979)). In identifying the optimal type of antibody for a particularapplication, the effector functions of the antibodies can be taken intoaccount. For example, hIgG1 antibodies have a relatively long half life,are very effective at fixing complement, and they bind to both FcγRI andFcγRII. In contrast, human IgG4 antibodies have a shorter half life, donot fix complement and have a lower affinity for the FcRs. Replacementof serine 228 with a proline (S228P) in the Fc region of IgG4 reducesheterogeneity observed with hIgG4 and extends the serum half life(Kabat, et al., “Sequences of proteins of immunological interest”5.sup.th Edition (1991); Angal, et al., Mol Immunol 30(1): 105-8(1993)). A second mutation that replaces leucine 235 with a glutamicacid (L235E) eliminates the residual FcR binding and complement bindingactivities (Alegre, et al., J Immunol 148(11): 3461-8 (1992)). Theresulting antibody with both mutations is referred to as IgG4PE. Thenumbering of the hIgG4 amino acids was derived from EU numberingreference: Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969). PMID: 5257969. In one embodiment of the present invention ICOSantigen binding proteins comprising an IgG4 Fc region comprising thereplacement S228P and L235E may have the designation IgG4PE. Thus, anICOS binding protein having the heavy chain variable region H2 and thelight chain variable region L5 and an IgG4PE Fc region will bedesignated as H2L5 IgG4PE or synonymously as H2L5 hIgG4PE.

Enhanced ADCC/CDC

As is understood in the art various techniques are known which willincrease the ADCC and/or the CDC activity of an antibody. These include,but are not limited to, various mutation in the Fc region, Complegentand Potelligent technologies. In one aspect of the present invention oneor more ADCC/CDC enhancing techniques may be applied to the ICOS bindingproteins of the present invention.

Mutation

Human IgG1 constant regions containing specific mutations or alteredglycosylation on residue Asn297 have also been described to enhancebinding to Fc receptors. In some cases these mutations have also beenshown to enhance ADCC and CDC, see for example, Kellner (2013).

In one embodiment of the present invention, such mutations are in one ormore of positions selected from 239, 332 and 330 (IgG1), or theequivalent positions in other IgG isotypes. Examples of suitablemutations are S239D and I332E and A330L. In one embodiment the antigenbinding protein of the invention herein described is mutated atpositions 239 and 332, for example S239D and I332E or in a furtherembodiment it is mutated at three or more positions selected from 239and 332 and 330, for example S239D and I332E and A330L (EU indexnumbering).

Complegent

In one embodiment of the present invention there is provided an antigenbinding protein comprising a chimeric heavy chain constant region forexample an antigen binding protein comprising a chimeric heavy chainconstant region with at least one CH2 domain from IgG3 such that theantigen binding protein has enhanced effector function, for examplewherein it has enhanced ADCC or enhanced CDC, or enhanced ADCC and CDCfunctions. In one such embodiment, the antigen binding protein maycomprise one CH2 domain from IgG3 or both CH2 domains may be from IgG3.

Also provided is a method of producing an antigen binding proteinaccording to the invention comprising the steps of:

a) culturing a recombinant host cell comprising an expression vectorcomprising an isolated nucleic acid as described herein wherein theexpression vector comprises a nucleic acid sequence encoding an Fcdomain having both IgG1 and IgG3 Fc domain amino acid residues; andb) recovering the antigen binding protein.

Such methods for the production of antigen binding proteins can beperformed, for example, using the COMPLEGENT™ technology systemavailable from BioWa, Inc. (Princeton, N.J.) and Kyowa Hakko Kogyo (now,Kyowa Hakko Kirin Co., Ltd.) Co., Ltd. In which a recombinant host cellcomprising an expression vector in which a nucleic acid sequenceencoding a chimeric Fc domain having both IgG1 and IgG3 Fc domain aminoacid residues is expressed to produce an antigen binding protein havingenhanced complement dependent cytotoxicity (CDC) activity that isincreased relative to an otherwise identical antigen binding proteinlacking such a chimeric Fc domain. Aspects of the COMPLEGENT™ technologysystem are described in WO2007011041 and US20070148165 each of which areincorporated herein by reference. In an alternative embodiment CDCactivity may be increased by introducing sequence specific mutationsinto the Fc region of an IgG chain. Those of ordinary skill in the artwill also recognize other appropriate systems.

Potelligent

The present invention also provides a method for the production of anantigen binding protein according to the invention comprising the stepsof:

a) culturing a recombinant host cell comprising an expression vectorcomprising the isolated nucleic acid as described herein, wherein theFUT8 gene encoding alpha-1,6-fucosyltransferase has been inactivated inthe recombinant host cell; andb) recovering the antigen binding protein.

Such methods for the production of antigen binding proteins can beperformed, for example, using the POTELLIGENT™ technology systemavailable from BioWa, Inc. (Princeton, N.J.) in which CHOK1SV cellslacking a functional copy of the FUT8 gene produce monoclonal antibodieshaving enhanced antibody dependent cell mediated cytotoxicity (ADCC)activity that is increased relative to an identical monoclonal antibodyproduced in a cell with a functional FUT8 gene. Aspects of thePOTELLIGENT™ technology system are described in U.S. Pat. No. 7,214,775,U.S. Pat. No. 6,946,292, WO0061739 and WO0231240 all of which areincorporated herein by reference. Those of ordinary skill in the artwill also recognize other appropriate systems.

It will be apparent to those skilled in the art that such modificationsmay not only be used alone but may be used in combination with eachother in order to further enhance effector function.

In one such embodiment of the present invention there is provided anantigen binding protein comprising a heavy chain constant region whichcomprises a mutated and chimaeric heavy chain constant region forexample wherein an antigen binding protein comprising at least one CH2domain from IgG3 and one CH2 domain from IgG1, wherein the IgG1 CH2domain has one or more mutations at positions selected from 239 and 332and 330 (for example the mutations may be selected from S239D and 1332Eand A330L) such that the antigen binding protein has enhanced effectorfunction, for example wherein it has one or more of the followingfunctions, enhanced ADCC or enhanced CDC, for example wherein it hasenhanced ADCC and enhanced CDC. In one embodiment the IgG1 CH2 domainhas the mutations S239D and 1332E.

In an alternative embodiment of the present invention there is providedan antigen binding protein comprising a chimaeric heavy chain constantregion and which has an altered glycosylation profile. In one suchembodiment the heavy chain constant region comprises at least one CH2domain from IgG3 and one CH2 domain from IgG1 and has an alteredglycosylation profile such that the ratio of fucose to mannose is 0.8:3or less, for example wherein the antigen binding protein isdefucosylated so that said antigen binding protein has an enhancedeffector function in comparison with an equivalent antigen bindingprotein with an immunoglobulin heavy chain constant region lacking saidmutations and altered glycosylation profile, for example wherein it hasone or more of the following functions, enhanced ADCC or enhanced CDC,for example wherein it has enhanced ADCC and enhanced CDC

In an alternative embodiment the antigen binding protein has at leastone IgG3 CH2 domain and at least one heavy chain constant domain fromIgG1 wherein both IgG CH2 domains are mutated in accordance with thelimitations described herein.

In one aspect of the invention there is provided a method of producingan antigen binding protein according to the invention described hereincomprising the steps of:

a) culturing a recombinant host cell containing an expression vectorcontaining an isolated nucleic acid as described herein, said expressionvector further comprising a Fc nucleic acid sequence encoding a chimericFc domain having both IgG1 and IgG3 Fc domain amino acid residues, andwherein the FUT8 gene encoding alpha-1,6-fucosyltransferase has beeninactivated in the recombinant host cell; andb) recovering the antigen binding protein.

Such methods for the production of antigen binding proteins can beperformed, for example, using the ACCRETAMAB™ technology systemavailable from BioWa, Inc. (Princeton, N.J.) which combines thePOTELLIGENT™ and COMPLEGENT™ technology systems to produce an antigenbinding protein having both ADCC and CDC enhanced activity that isincreased relative to an otherwise identical monoclonal antibody lackinga chimeric Fc domain and which has fucose on the oligosaccharide

In yet another embodiment of the present invention there is provided anantigen binding protein comprising a mutated and chimeric heavy chainconstant region wherein said antigen binding protein has an alteredglycosylation profile such that the antigen binding protein has enhancedeffector function, for example wherein it has one or more of thefollowing functions, enhanced ADCC or enhanced CDC. In one embodimentthe mutations are selected from positions 239 and 332 and 330, forexample the mutations are selected from S239D and I332E and A330L. In afurther embodiment the heavy chain constant region comprises at leastone CH2 domain from IgG3 and one Ch2 domain from IgG1. In one embodimentthe heavy chain constant region has an altered glycosylation profilesuch that the ratio of fucose to mannose is 0.8:3 or less for examplethe antigen binding protein is defucosylated, so that said antigenbinding protein has an enhanced effector function in comparison with anequivalent non-chimaeric antigen binding protein or with animmunoglobulin heavy chain constant region lacking said mutations andaltered glycosylation profile.

The long half-life of IgG antibodies is reported to be dependent on itsbinding to FcRn. Therefore, substitutions that increase the bindingaffinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence ofthe interaction by engineering the constant region have been extensivelystudied Kuo and Aveson (2011).

Another means of modifying antigen binding proteins of the presentinvention involves increasing the in-vivo half life of such proteins bymodification of the immunoglobulin constant domain or FcRn (Fc receptorneonate) binding domain.

In adult mammals, FcRn, also known as the neonatal Fc receptor, plays akey role in maintaining serum antibody levels by acting as a protectivereceptor that binds and salvages antibodies of the IgG isotype fromdegradation. IgG molecules are endocytosed by endothelial cells, and ifthey bind to FcRn, are recycled out into circulation. In contrast, IgGmolecules that do not bind to FcRn enter the cells and are targeted tothe lysosomal pathway where they are degraded.

The neonatal FcRn receptor is believed to be involved in both antibodyclearance and the transcytosis across tissues, Kuo and Aveson, (2011).Human IgG1 residues determined to interact directly with human FcRnincludes Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.Switches at any of these positions described in this section may enableincreased serum half-life and/or altered effector properties of antigenbinding proteins of the invention.

Mutations to Increase Half Life by Increasing Affinity to FcRn

Antigen binding proteins of the present invention may have one or moreamino acid modifications that increase the affinity of the constantdomain or fragment thereof for FcRn. These may result in increasedhalf-life of these proteins Kuo and Aveson (2011) Increasing thehalf-life of therapeutic and diagnostic IgG's and other bioactivemolecules has many benefits including reducing the amount and/orfrequency of dosing of these molecules. In one embodiment there istherefore provided an antigen binding according to the inventionprovided herein or a fusion protein comprising all or a portion (an FcRnbinding portion) of an IgG constant domain having one or more of theseamino acid modifications and a non-IgG protein or non-protein moleculeconjugated to such a modified IgG constant domain, wherein the presenceof the modified IgG constant domain increases the in vivo half life ofthe antigen binding protein.

A number of methods are known that can result in increased half-life(Kuo and Aveson, (2011)), including amino acid modifications may begenerated through techniques including alanine scanning mutagenesis,random mutagenesis and screening to assess the binding to FcRn and/orthe in vivo behaviour. Computational strategies followed by mutagenesismay also be used to select one of amino acid mutations to mutate.

The present invention therefore provides a variant of an antigen bindingprotein with optimized binding to FcRn. In a preferred embodiment, thesaid variant of an antigen binding protein comprises at least one aminoacid modification in the Fc region of said antigen binding protein,wherein said modification is selected from the group consisting of 226,227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259,264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294,297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320,322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380,382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398,399, 400, 401 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420,421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and447 of the Fc region as compared to said parent polypeptide, wherein thenumbering of the amino acids in the Fc region is that of the EU index inKabat.

In a further aspect of the invention the modifications areM252Y/S254T/T256E.

Additionally, various publications describe methods for obtainingphysiologically active molecules whose half-lives are modified, see forexample Kontermann (2009) either by introducing an FcRn-bindingpolypeptide into the molecules or by fusing the molecules withantibodies whose FcRn-binding affinities are preserved but affinitiesfor other Fc receptors have been greatly reduced or fusing with FcRnbinding domains of antibodies.

pH Switch Technology to Increase Half Life

Although substitutions in the constant region are able to significantlyimprove the functions of therapeutic IgG antibodies, substitutions inthe strictly conserved constant region have the risk of immunogenicityin human and substitution in the highly diverse variable region sequencemight be less immunogenic. Reports concerned with the variable regioninclude engineering the CDR residues to improve binding affinity to theantigen and engineering, the CDR and framework residues to improvestability and decrease immunogenicity risk. As is known, improvedaffinity to the antigen can be achieved by affinity maturation using thephage or ribosome display of a randomized library.

Improved stability can be rationally obtained from sequence- andstructure-based rational design. Decreased immunogenicity risk(deimmunization) can be accomplished by various humanizationmethodologies and the removal of T-cell epitopes, which can be predictedusing in silico technologies or determined by in vitro assays.Additionally, variable regions have been engineered to lower pI. Alonger half life was observed for these antibodies as compared to wildtype antibodies despite comparable FcRn binding. Engineering orselecting antibodies with pH dependent antigen binding to modifyantibody and/or antigen half life eg IgG2 antibody half life can beshortened if antigen-mediated clearance mechanisms normally degrade theantibody when bound to the antigen. Similarly, the antigen:antibodycomplex can impact the half-life of the antigen, either extendinghalf-life by protecting the antigen from the typical degradationprocesses, or shortening the half-life via antibody-mediateddegradation. One embodiment relates to antibodies with higher affinityfor antigen at pH 7.4 as compared to endosomal pH (i.e., pH 5.5-6.0)such that the KD ratio at pH5.5/pH 7.4 or at pH 6.0/pH 7.4 is 2 or more.For example to enhance the pharmacokinetic (PK) and pharmacodynamic (PD)properties of the antibody, it is possible to engineer pH-sensitivebinding to the antibody by introducing histidines into CDR residues.

Additionally, methods of producing an antigen binding protein with adecreased biological half-life are also provided. A variant IgG in whichHis435 is mutated to alanine results in the selective loss of FcRnbinding and a significantly reduced serum half-life (see for exampleU.S. Pat. No. 6,165,745 discloses a method of producing an antigenbinding protein with a decreased biological half-life by introducing amutation into the DNA segment encoding the antigen binding protein. Themutation includes an amino acid substitution at position 253, 310, 311,433, or 434 of the Fc-hinge domain.

Linkers

Protein scaffolds may be the same as naturally occurring sequences, suchas Ig sequences, or be fragments of naturally occurring sequences, andmay contain additional sequences which may be naturally occurring, froma difference source or synthetic, and which may be added at the N or Cterminus of the scaffold. Such additional sequences may be considered tobe linkers when they link an epitope binding domain and proteinscaffold, such as those defined herein.

In another aspect the antigen binding construct consists of, or consistsessentially of, an Fc region of an antibody, or a part thereof, linkedat each end, directly or indirectly (for example, via a linker sequence)to an epitope binding domain. Such an antigen binding construct maycomprise 2 epitope-binding domains separated by an Fc region, or partthereof. By separated is meant that the epitope-binding domains are notdirectly linked to one another, and in one aspect are located atopposite ends (C and N terminus) of an Fc region, or any other scaffoldregion.

In one aspect the antigen binding construct comprises 2 scaffold regionseach bound to 2 epitope binding domains, for example at the N and Ctermini of each scaffold region, either directly or indirectly via alinker.

Protein scaffolds of the present invention may be linked toepitope-binding domains by the use of linkers. Examples of suitablelinkers include amino acid sequences which may be from 1 amino acid to150 amino acids in length, or from 1 amino acid to 140 amino acids, forexample, from 1 amino acid to 130 amino acids, or from 1 to 120 aminoacids, or from 1 to 80 amino acids, or from 1 to 50 amino acids, or from1 to 20 amino acids, or from 1 to 10 amino acids, or from 5 to 18 aminoacids. Such sequences may have their own tertiary structure, forexample, a linker of the present invention may comprise a singlevariable domain. The size of a linker in one embodiment is equivalent toa single variable domain. Suitable linkers may be of a size from 1 to100 angstroms, for example may be of a size from 20 to 80 angstroms orfor example may be of a size from 20 to 60 angstroms or for example lessthan 40 angstroms, or less than 20 angstroms, or less than 5 angstromsin length.

In one embodiment of the present invention, ICOS binding proteins areprovided comprising one or more of: CDRH1 as set forth in SEQ ID NO:1;CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3;CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO:5and/or CDRL3 as set forth in SEQ ID NO:6 or a direct equivalent of eachCDR wherein a direct equivalent has no more than two amino acidsubstitutions in said CDR.

In one embodiment of the present invention, ICOS binding proteins areprovided which specifically binds to human ICOS comprising a V_(H)domain comprising an amino acid sequence at least 90% identical to theamino acid sequence set forth in SEQ ID NO:7 and/or a V_(L) domaincomprising an amino acid sequence at least 90% identical to the aminoacid sequence set forth in SEQ ID NO:8. In one aspect, the ICOS bindingproteins of the present invention, also bind to cynomolgus ICOS. In oneaspect, they do not bind to murine ICOS.

In one embodiment, the ICOS binding proteins of the invention are ICOSagonists. In one aspect, the ICOS binding proteins increase IFN-gammaproduction in the presence of Tcells. In another aspect, the ICOSbinding proteins of the present invention stimulate Tcell proliferation.

In one embodiment, the ICOS binding protein of the invention bind tohuman ICOS with

-   -   an association rate constant (kon) of at least 1×10⁵ M-1s-1; and        a dissociation rate constant (koff) of less than 6×10-5 s-1; or    -   a dissociation constant (Kd) of less than 100 nM

wherein the high affinity is measured by Biacore.

In one embodiment the ICOS binding protein comprises CDRH3 (SEQ ID NO:3)or a variant of SEQ ID NO. 3. In another embodiment the ICOS bindingproteins comprise one or more of: CDRH1 (SEQ ID NO:1); CDRH2 (SEQ IDNO:2); CDRH3 (SEQ ID NO:3); CDRL1 (SEQ ID NO:4); CDRL2 (SEQ ID NO:5);and/or CDRL3 (SEQ ID NO:6). In one embodiment, the ICOS binding proteinscomprise heavy chain CDRs as set forth in SEQ ID NO:1; SEQ ID NO:2; andSEQ ID NO:3 and light chain CDRs as set forth in SEQ ID NO:4; SEQ IDNO:5; and SEQ ID NO:6.

In one embodiment, the ICOS binding proteins comprise a V_(H) domainhaving 90% sequence identity to the amino acid sequence set forth in SEQID NO:7; and a V_(L) domain having 90% sequence identity to the aminoacid sequence shown in the amino acid sequence set forth in SEQ ID NO:8.In one aspect, the ICOS binding proteins comprise a V_(H) domain havingthe amino acid sequence set forth in SEQ ID NO. 7 and the V_(L) domaincomprising the amino acid sequence as set forth in SEQ ID NO:8. In oneaspect, the ICOS binding proteins comprise a heavy chain variable domainconsisting of SEQ ID NO:7. In one aspect, the ICOS binding proteincomprises a light chain variable domain consisting of SEQ ID NO:8.

In one embodiment, the invention provides an ICOS binding protein orantigen binding portion thereof comprising a V_(H) domain comprising anamino acid sequence at least 90% identical to the amino acid sequenceset forth in SEQ ID NO:7; and a V_(L) domain comprising an amino acidsequence at least 90% identical to the amino acid sequence as set forthin SEQ ID NO:8 wherein said ICOS binding protein or antigen bindingportion thereof specifically binds to human ICOS. In one embodiment, theICOS binding protein or antigen binding portion thereof comprising aV_(H) domain comprising an amino acid sequence at least 90% identical tothe amino acid sequence set forth in SEQ ID NO:7; and a V_(L) domaincomprising an amino acid sequence at least 90% identical to the aminoacid sequence as set forth in SEQ ID NO:8 wherein said ICOS bindingprotein or antigen binding portion thereof specifically binds to humanICOS further comprises heavy chain CDRs having the amino acid sequencesset forth in SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chainCDRs having the amino acid sequences set forth in SEQ ID NO:4; SEQ IDNO:5; and SEQ ID NO:6. In one aspect, the ICOS binding protein orantigen binding portion thereof comprises a V_(H) domain comprising anamino acid sequence set forth in SEQ ID NO:7; and a V_(L) domaincomprises the amino acid sequence set forth in SEQ ID NO:8. In oneembodiment, the ICOS binding protein or antigen binding portion thereofis an agonist to human ICOS. In one embodiment the ICOS binding proteinor antigen binding portion thereof further comprising an IgG4 isotypescaffold or a variant thereof. In one embodiment, the ICOS bindingprotein or antigen binding portion thereof comprises a hIgG4PE scaffold.

In one embodiment, the ICOS binding protein of the present invention isa humanized monoclonal antibody comprising a heavy chain amino acidsequence having at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to the amino acid sequence set forth inSEQ ID NO:23.

(SEQ ID NO: 23) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWMGLISIYSDHTNYNQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

In one embodiment, the ICOS binding protein of the present invention isa humanized monoclonal antibody comprising a light chain amino acidsequence having at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to the amino acid sequence set forth inSEQ ID NO:24.

(SEQ ID NO: 24) EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC

In one embodiment, the ICOS binding protein of the present invention isa humanized monoclonal antibody comprising the heavy chain amino acidsequence set forth in SEQ ID NO:23 and the light chain amino acidsequence set forth in SEQ ID NO:24. In one embodiment, the ICOS bindingprotein of the present invention further comprises an hIgGPE scaffold.

In one embodiment, the ICOS binding protein or antigen binding portionthereof wherein said ICOS binding protein is a humanized monoclonalantibody. Also provided by the present invention are pharmaceuticalcompositions comprising the ICOS binding protein or antigen bindingportion thereof of claim and a pharmaceutically acceptable carrier.

The present invention provides methods of treating a disease selectedfrom cancer, infectious disease, and/or sepsis in a human in needthereof which method comprises the step of administering apharmaceutical composition of the present invention said human. In oneaspect, the method further comprises administering at least oneanti-neoplastic agent, at least one second immune-modulatory agent,and/or at least one immunostimulatory adjuvant to said human. In oneaspect, the second immuno-modulatory agent is selected from: ananti-CTLA4 antibody, and anti-PD-1 antibody, an anti-PDL1 antibody andan anti OX40 antibody. In one aspect the anti-CTLA4 antibody isipilimumab. In one aspect the anti-PD-1 antibody is selected frompembrolizumab and/or nivolumab.

In one embodiment of the present invention, methods are provided fortreating cancer in a human comprising administering a therapeuticallyacceptable amount of the ICOS binding protein or antigen binding portionthereof to a human. In some aspects the cancer is selected fromcolorectal cancer (CRC), esophageal, cervical, bladder, breast, head andneck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell,non-small cell lung carcinoma, mesothelioma, and prostate cancer.

In one embodiment, methods are provided for treating infectious diseasein a human comprising administering a therapeutically acceptable amountof the ICOS binding protein or antigen binding portion thereof to ahuman. In one aspect, the infectious diseases is HIV.

In one embodiment, methods are provided for treating sepsis in a humancomprising administering a therapeutically acceptable amount of the ICOSbinding protein or antigen binding portion thereof to a human.

In one embodiment, methods are provided for stimulating T cellproliferation, inducing T cell activation and/or inducing cytokineproduction in a human comprising administering a pharmaceuticalcomposition of the invention to said human.

The present invention also provides polynucleotides encoding the ICOSbinding protein or antigen binding portion thereof of the presentinvention. In one embodiment, host cells are provided comprisingpolynucleotides encoding the ICOS binding proteins or antigen bindingportions thereof of the present invention. The present invention alsoprovides methods of making an ICOS binding protein or antigen bindingportion thereof comprising the steps of a) culturing host cellcomprising a polynucleotide encoding an ICOS binding protein or antigenbinding portion thereof of the present invention under suitableconditions to express said ICOS binding protein or antigen bindingportion thereof; and b) isolating said ICOS binding protein or antigenbinding portion thereof.

The present invention provides an isolated humanized monoclonal antibodycomprising a V_(H) domain comprising an amino acid sequence set forth inSEQ ID NO:7; a V_(L) domain comprising an amino acid sequence set forthin SEQ ID NO:8; and a hIgG4 scaffold or a variant thereof. In oneaspect, the hIgG4 scaffold is hIgG4PE.

In one embodiment, ICOS binding proteins or antigen binding portionsthereof are provided, wherein the ICOS binding protein or antigenbinding portion thereof cross-competes for binding for human ICOS with areference antibody or antigen binding portion thereof comprising a V_(H)domain comprising an amino acid sequence set forth in SEQ ID NO:7; and aV_(L) domain comprising the amino acid sequence set forth in SEQ IDNO:8.

In one embodiment, the ICOS binding proteins of the invention stimulateT cell proliferation when placed in contact with a T cell. In oneembodiment, the ICOS binding proteins of the invention induce T cellactivation when placed in contact with a T cell. T cell activation canbe measured by an increase in percent expression levels of certainactivation markers such as, but not limited to, CD69, CD25, and/or OX40.In one embodiment, the ICOS binding proteins of the present inventionstimulate cytokine production when placed in contact with a T cell. TheICOS binding proteins bind to human FcγRIIb but do not bind to humanFcγRIIa or human FcγRIIIa. Additionally, the ICOS binding proteinssuitably do not deplete ICOS expressing T cells when contacted with ICOSexpressing T cells. In some aspects, the ICOS binding proteinscross-link a T cell with a second cell when contacted with said T cellin the presence of a second cell. This cross-linking can occur throughengagement of the ICOS binding protein with a FcγR on the second cell.FcγR expressing cells include, but are not limited to monocytes, Blymphocytes, follicular dendritic cells, natural killer cells,macrophages, neutrophils, eosinophils, basophils, and mast cells. Thus,in one embodiment the ICOS binding proteins can be administered to amammal wherein the ICOS binding protein will act as an agonist to ICOSon a cell and will also engage an FcγR on a second cell.

In one embodiment, the ICOS binding proteins comprise a scaffoldselected from human IgG1 isotype or variant thereof and human IgG4isotype or variant thereof. Suitably, the scaffold comprises a humanIgG4 isotype scaffold or variant thereof. In one aspect, the scaffoldcomprises a hIgG4PE scaffold.

In one embodiment, the ICOS binding protein is a monoclonal antibody.Suitably the ICOS binding protein is a humanized monoclonal antibody. Inone aspect the monoclonal antibodies of the present invention can befully human.

In another aspect, the ICOS binding protein is a fragment which is aFab, Fab′, F(ab′)₂, Fv, diabody, triabody, tetrabody, miniantibody,minibody, isolated V_(H) or isolated V_(L). In one embodiment, the ICOSbinding protein is an antigen binding portion thereof.

In some aspects the ICOS binding protein binds to human ICOS with anaffinity of stronger than 0.6 nM. In one aspect, the affinity is 100 nMor stronger. In one embodiment the ICOS binding protein has a KD of 100nM for ICOS. Suitably, the KD of the ICOS binding protein for ICOS is100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 2 nM orless or 1 nM or less.

In one embodiment, the present invention provides humanized monoclonalantibodies that are agonists to human ICOS. In one embodiment, thepresent invention provides humanized monoclonal antibodies comprisingheavy chain variable region CDRs having the amino acid sequences setforth in SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chainvariable region CDRS having the amino acid sequences set forth in SEQ IDNO:4; SEQ ID NO:5; and SEQ ID NO:6. In one aspect, the humanizedmonoclonal antibody is able to stimulate cytokine production and/or Tcell proliferation when contacted with a T cell while not inducingcomplement, ADCC or CDC. In one embodiment, the humanized monoclonalantibody has variant human IgG1 Fc region. In one embodiment, thehumanized monoclonal antibody has a human IgG4 Fc region or variantthereof. In one embodiment, the humanized monoclonal antibody has ahIgG4PE Fc region. In one aspect, the humanized monoclonal antibodycomprises a V_(H) domain comprising an amino acid sequence at least 90%identical to the amino acid sequence as set forth in SEQ ID NO:7; and aV_(L) domain comprising an amino acid sequence at least 90% identical tothe amino acid sequence as set forth in SEQ ID NO:8. In one aspect thehumanized monoclonal antibody comprises a V_(H) domain comprising anamino acid sequence set forth in SEQ ID NO:7; and a V_(L) domaincomprising the amino acid sequence as set forth in SEQ ID NO:8. In oneaspect the humanized monoclonal antibody comprises and hIgG4PE scaffold.Furthermore, humanized monoclonal antibodies of the present inventionare shown to stimulate T cell proliferation when contacted with a CD4+or a CD8+ T cell. Humanized monoclonal antibodies of the presentinvention are shown to induce T cell activation and stimulate cytokineproduction.

In one embodiment, the humanized monoclonal antibody comprises anhIgG4PE scaffold and comprises a V_(H) domain comprising an amino acidsequence set forth in SEQ ID NO:7 and a V_(L) domain comprising an aminoacid sequence set forth in SEQ ID NO:8. The antibodies of the presentinvention may stimulate cytokine production when contacted with a Tcell.

In one embodiment, an ICOS binding protein is provided that competes forICOS binding with any one of the ICOS binding proteins of the invention.As is understood in the art and described herein, binding competitioncan be measured by comparing competition for ligand binding to ICOS inthe presence of one or more ICOS binding proteins. As is also understoodin the art, ICOS is expressed on CD4+ and CD8+ T cells as well as Tregcells. The ICOS binding proteins of the present invention act as agonistto ICOS on T cells. They also act to block the interaction betweenICOS-L and ICOS expressed on both T cells and Treg cells. Thus, in oneembodiment, methods are provided to block the interaction of ICOS-L withICOS on Treg cells. ICOS expressing Treg cells can be found in varioustypes of tumors including liquid tumors such as lymphoma. Thus, in oneaspect of the present invention, methods of treating a cancer areprovided comprising administering an ICOS binding protein of theinvention wherein the ICOS binding protein blocks the interaction ofICOS-L with ICOS on Treg cells.

Further to the invention, are pharmaceutical compositions comprising anICOS binding protein or a monoclonal antibody described herein. In oneaspect the pharmaceutical composition of the present invention furthercomprise at least one anti-neoplastic agent. In one aspect thepharmaceutical composition of the present invention further comprise atleast one second immunomodulatory agent. In one aspect, thepharmaceutical composition of the present invention further comprisingat least one immunostimulatory adjuvant.

In one embodiment, methods are provided for treating cancer and/orinfectious disease in a human in need thereof wherein said methodcomprises the step of administering a pharmaceutical composition of theinvention to said human. In one embodiment the human has cancer. In oneembodiment the human has an infectious disease. In one embodiment thehuman has HIV. In one aspect the method further comprises administeringat least one anti-neoplastic agent to said human. In another aspect themethod further comprises administering at least one secondimmune-modulatory agent to said human. In yet another aspect the methodfurther comprises administering an immunostimulatory adjuvant to saidhuman.

In one aspect the human has a solid tumor. In one aspect the tumor isselected from head and neck cancer, gastric cancer, melanoma, renal cellcarcinoma (RCC), esophageal cancer, non-small cell lung carcinoma,prostate cancer, colorectal cancer, ovarian cancer and pancreaticcancer. In one aspect the human has one or more of the following:colorectal cancer (CRC), esophageal, cervical, bladder, breast, head andneck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell,non-small cell lung carcinoma, mesothelioma, and prostate cancer. Inanother aspect the human has a liquid tumor such as diffuse large B celllymphoma (DLBCL), multiple myeloma, chronic lyphomblastic leukemia(CLL), follicular lymphoma, acute myeloid leukemia and chronicmyelogenous leukemia.

The present disclosure also relates to a method for treating orlessening the severity of a cancer selected from: brain (gliomas),glioblastomas, Bannayan-Zonana syndrome, Cowden disease,Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm'stumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma,colon, head and neck, kidney, lung, liver, melanoma, ovarian,pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone,thyroid, lymphoblastic T-cell leukemia, chronic myelogenous leukemia,chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblasticleukemia, acute myelogenous leukemia, chronic neutrophilic leukemia,acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic largecell leukemia, mantle cell leukemia, multiple myeloma megakaryoblasticleukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocyticleukemia, erythroleukemia, malignant lymphoma, Hodgkins lymphoma,non-hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt'slymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelialcancer, lung cancer, vulval cancer, cervical cancer, endometrial cancer,renal cancer, mesothelioma, esophageal cancer, salivary gland cancer,hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccalcancer, cancer of the mouth, GIST (gastrointestinal stromal tumor) andtesticular cancer.

By the term “treating” and grammatical variations thereof as usedherein, is meant therapeutic therapy. In reference to a particularcondition, treating means: (1) to ameliorate or prevent the condition ofone or more of the biological manifestations of the condition, (2) tointerfere with (a) one or more points in the biological cascade thatleads to or is responsible for the condition or (b) one or more of thebiological manifestations of the condition, (3) to alleviate one or moreof the symptoms, effects or side effects associated with the conditionor treatment thereof, (4) to slow the progression of the condition orone or more of the biological manifestations of the condition and/or (5)to cure said condition or one or more of the biological manifestationsof the condition by eliminating or reducing to undetectable levels oneor more of the biological manifestations of the condition for a periodof time considered to be a state of remission for that manifestationwithout additional treatment over the period of remission. One skilledin the art will understand the duration of time considered to beremission for a particular disease or condition. Prophylactic therapy isalso contemplated thereby. The skilled artisan will appreciate that“prevention” is not an absolute term. In medicine, “prevention” isunderstood to refer to the prophylactic administration of a drug tosubstantially diminish the likelihood or severity of a condition orbiological manifestation thereof, or to delay the onset of suchcondition or biological manifestation thereof. Prophylactic therapy isappropriate, for example, when a subject is considered at high risk fordeveloping cancer, such as when a subject has a strong family history ofcancer or when a subject has been exposed to a carcinogen.

As used herein, the terms “cancer,” “neoplasm,” and “tumor” are usedinterchangeably and, in either the singular or plural form, refer tocells that have undergone a malignant transformation that makes thempathological to the host organism. Primary cancer cells can be readilydistinguished from non-cancerous cells by well-established techniques,particularly histological examination. The definition of a cancer cell,as used herein, includes not only a primary cancer cell, but any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by procedures such as computed tomography(CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound orpalpation on physical examination, and/or which is detectable because ofthe expression of one or more cancer-specific antigens in a sampleobtainable from a patient. Tumors may be a hematopoietic (or hematologicor hematological or blood-related) cancer, for example, cancers derivedfrom blood cells or immune cells, which may be referred to as “liquidtumors.” Specific examples of clinical conditions based on hematologictumors include leukemias such as chronic myelocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia and acute lymphocyticleukemia; plasma cell malignancies such as multiple myeloma, MGUS andWaldenstrom's macroglobulinemia; lymphomas such as non-Hodgkin'slymphoma, Hodgkin's lymphoma; and the like.

The cancer may be any cancer in which an abnormal number of blast cellsor unwanted cell proliferation is present or that is diagnosed as ahematological cancer, including both lymphoid and myeloid malignancies.Myeloid malignancies include, but are not limited to, acute myeloid (ormyelocytic or myelogenous or myeloblastic) leukemia (undifferentiated ordifferentiated), acute promyeloid (or promyelocytic or promyelogenous orpromyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic)leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia andmegakaryocytic (or megakaryoblastic) leukemia. These leukemias may bereferred together as acute myeloid (or myelocytic or myelogenous)leukemia (AML). Myeloid malignancies also include myeloproliferativedisorders (MPD) which include, but are not limited to, chronicmyelogenous (or myeloid) leukemia (CIVIL), chronic myelomonocyticleukemia (CMML), essential thrombocythemia (or thrombocytosis), andpolcythemia vera (PCV). Myeloid malignancies also include myelodysplasia(or myelodysplastic syndrome or MDS), which may be referred to asrefractory anemia (RA), refractory anemia with excess blasts (RAEB), andrefractory anemia with excess blasts in transformation (RAEBT); as wellas myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

Hematopoietic cancers also include lymphoid malignancies, which mayaffect the lymph nodes, spleens, bone marrow, peripheral blood, and/orextranodal sites. Lymphoid cancers include B-cell malignancies, whichinclude, but are not limited to, B-cell non-Hodgkin's lymphomas(B-NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (oraggressive) or high-grade (very aggressive). Indolent Bcell lymphomasinclude follicular lymphoma (FL); small lymphocytic lymphoma (SLL);marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL,splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacyticlymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT orextranodal marginal zone) lymphoma. Intermediate-grade B-NHLs includemantle cell lymphoma (MCL) with or without leukemic involvement, diffuselarge cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLsinclude Burkitt's lymphoma (BL), Burkitt-like lymphoma, smallnon-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. OtherB-NHLs include immunoblastic lymphoma (or immunocytoma), primaryeffusion lymphoma, HIV associated (or AIDS related) lymphomas, andpost-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cellmalignancies also include, but are not limited to, chronic lymphocyticleukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom'smacroglobulinemia (WM), hairy cell leukemia (HCL), large granularlymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic orlymphoblastic) leukemia, and Castleman's disease. NHL may also includeT-cell non-Hodgkin's lymphoma s(T-NHLs), which include, but are notlimited to T-cell non-Hodgkin's lymphoma not otherwise specified (NOS),peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma(ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal naturalkiller (NK) cell/T-cell lymphoma, gamma/delta lymphoma, cutaneous T celllymphoma, mycosis fungoides, and Sezary syndrome.

Hematopoietic cancers also include Hodgkin's lymphoma (or disease)including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin'slymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant(LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocytedepleted Hodgkin's lymphoma. Hematopoietic cancers also include plasmacell diseases or cancers such as multiple myeloma (MM) includingsmoldering MM, monoclonal gammopathy of undetermined (or unknown orunclear) significance (MGUS), plasmacytoma (bone, extramedullary),lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia,plasma cell leukemia, and primary amyloidosis (AL). Hematopoieticcancers may also include other cancers of additional hematopoieticcells, including polymorphonuclear leukocytes (or neutrophils),basophils, eosinophils, dendritic cells, platelets, erythrocytes andnatural killer cells. Tissues which include hematopoietic cells referredherein to as “hematopoietic cell tissues” include bone marrow;peripheral blood; thymus; and peripheral lymphoid tissues, such asspleen, lymph nodes, lymphoid tissues associated with mucosa (such asthe gut-associated lymphoid tissues), tonsils, Peyer's patches andappendix, and lymphoid tissues associated with other mucosa, forexample, the bronchial linings.

The ICOS binding proteins, antibodies and antigen binding fragments ofthe invention can also be used to cure, prevent or treat infections andinfectious disease. The ICOS binding proteins can be used alone, or incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. The ICOS binding proteins of thepresent invention can be used to stimulate immune response to virusesinfectious to humans, such as, but not limited to, humanimmunodeficiency viruses, hepatitis viruses class A, B and C, EppsteinBarr virus, human cytomegalovirus, human papilloma viruses, herpesviruses. The ICOS binding proteins of the present invention can be usedto stimulate immune response to infection with bacterial or fungalparasites, and other pathogens. Suitably, the present invention providesmethods for treating humans that have been exposed to particular toxinsor pathogens. Accordingly, another aspect of the invention provides amethod of treating an infectious disease in a subject comprisingadministering to the subject ICOS binding protein, or antigen-bindingportion thereof.

Examples of infectious disease for which the ICOS binding proteins ofthe present invention may be useful include, but are not limited to,HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria,Leishmania, Staphylococcus aureus, Pseudomonas Aeruginosa. Some examplesof pathogenic viruses causing infections treatable by methods of theinvention include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV,HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measlesvirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, denguevirus, papillomavirus, molluscum virus, poliovirus, rabies virus, JCvirus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasiliensis.

Sepsis is a potentially deadly medical condition that is characterizedby a whole-body inflammatory state (called a systemic inflammatoryresponse syndrome or SIRS) and the presence of a known or suspectedinfection. The body may develop this inflammatory response by the immunesystem to microbes in the blood, urine, lungs, skin, or other tissues. Alay term for sepsis is blood poisoning, more aptly applied tosepticemia, below. Severe sepsis is the systemic inflammatory response,plus infection, plus the presence of organ dysfunction.

Septicemia is a related medical term referring to the presence ofpathogenic organisms in the bloodstream, leading to sepsis. The term hasnot been sharply defined.

Sepsis and cancer share similar immunosuppressive mechanisms includingincreased T regulatory cells, increased myeloid derived suppressorcells, increased expression of negative co-stimulatory molecules,decreased monocyte/macrophage HLA-DR expression. Sepsis and cancer areprototypical disorders of chronic inflammation. Chronic inflammationstimulates potent and sustained immunoregulatory responses, includingexpansion of T regulatory cells and up-regulation of PD-1 and othernegative regulators on effector cells. Barouch D. H. and Deeks S. G.;Immunologic strategies for HIV-1 remission and eradication. Science345:169-174 2014. Thus, in one aspect of the present inventions methodsare provided for treating sepsis in a human in need thereof comprisingadministering a therapeutically effective amount of an ICOS antigenbinding protein of the present invention to said human. Boomer, et al.JAMA 306:2594-2605 (2011); Meisel, et al. Granulocyte-macrophagecolony-stimulating factor to reverse sepsis-associatedimmunosuppression: a double-blind, randomized, placebo-controlledmulticenter trial. Am J Respir Crit Care Med 180:640-648 (2009); andHall, et al. Immunoparalysis and nosocomial infection in children withmultiple organ dysfunction syndrome. Intensive Care Med 37:525-532(2011).

The ICOS binding proteins of the invention can be used in conjunctionwith other recombinant proteins and/or peptides (such as tumor antigensor cancer cells) in order to increase an immune response to theseproteins (i.e., in a vaccination protocol).

For example, ICOS binding proteins thereof may be used to stimulateantigen-specific immune responses by co-administration of at least oneICOS binding protein with an antigen of interest (e.g., a vaccine).Accordingly, in another aspect the invention provides a method ofenhancing an immune response to an antigen in a subject, comprisingadministering to the subject: (i) the antigen; and (ii) an ICOS bindingprotein of the invention, such that an immune response to the antigen inthe subject is enhanced. The antigen can be, for example, a tumorantigen, a viral antigen, a bacterial antigen or an antigen from apathogen. Non-limiting examples of such antigens include, withoutlimitation, tumor antigens, or antigens from the viruses, bacteria orother pathogens.

A major hurdle to HIV eradication is the maintenance of latentlyinfected cells that do not express viral antigens, and escape immunesurveillance. Current strategies to eliminate the latent viral reservoirreferred to as the “kick and kill” strategy, aims to reactivate HIV geneexpression (“kick”) as cellular activation leads to HIV reactivation,and clear reactivated cells (“kill”). Cellular activation is governed bya balance of positive and negative regulators expressed on the surfaceof T cells. Altering this balance by agonising positive regulators andantagonising negative ones may facilitate HIV reactivation.

Inducible T cell Co-Stimulator (ICOS) is a positive regulator whoseexpression increases on CD4 T cells following stimulation. ICOSfunctions to promote T cell proliferation, cytokine production anddifferentiation. One important T cell subset that expresses high levelsof PD-1 and ICOS is T follicular helper cells (Tfh). Tfh cells help Bcells undergo differentiation, class switching, somatic hypermutationand are necessary for germinal center formation. Tfh cells aresignificantly expanded following HIV/SIV infection and theirdysregulation during chronic lentiviral infection contributes toimpaired B cell immunity. Sorted Tfh cells have been shown to containhigher levels of HIV DNA than other lymphoid CD4 subsets and virusoutgrowth is observed following stimulation. Tfh cells reside ingerminal centers and are exposed to HIV virions trapped on folliculardendritic cells that may facilitate their infection. In addition, CD8 Tcells have limited access to germinal centers and follicular CD8 cellsoften demonstrate reduced cytotoxicity, thus sparing Tfh cells fromantiviral surveillance. Thus, Tfh cells are an important protected HIVreservoir and strategies that target PD-1 and ICOS may selectivelytarget Tfh cells and have utility as part of an HIV cure regimen.Suitably, methods are provided for treating a human infected with HIVcomprising administering an ICOS binding protein or the antigen bindingportion thereof of the present invention.

As used herein “tumor antigens” are proteins that are produced by tumorcells that elicit an immune response, particularly T-cell mediatedimmune responses. The term “tumor antigen” as used herein includes bothtumor-specific antigens and tumor-associated antigens. Tumor-specificantigens are unique to tumor cells and do not occur on other cells inthe body. Tumor-associated antigens are not unique to a tumor cell andinstead is also expressed on a normal cell under conditions that fail toinduce a state of immunologic tolerance to the antigen. The expressionof the antigen on the tumor may occur under conditions that enable theimmune system to respond to the antigen. Tumor-associated antigens maybe antigens that are expressed on normal cells during fetal developmentwhen the immune system is immature and unable to respond or they may beantigens that are normally present at extremely low levels on normalcells but which are expressed at much higher levels on tumor cells.

Non-limiting examples of tumor antigens include the following:differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigenssuch, MAGE family antigens including but not limited to MAGE1, MAGES,MAGE10, MAGE11, MAGE12, MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA8, MAGEA9,MAGEB18, MAGEB6, MABEC1, MAGED2, MAGEE1, MAGEH1, MAGEL2, BAGE, GAGE-1,GAGE-2, p15; MEL4, melanoma associated antigen 100+, melanoma gp100,NRIP3, NYS48, OCIAD1, OFA-iLRP, OIP5, ovarian carcinoma-associatedantigen (OV632), PAGE4, PARP9, PATE, plastin L, PRAME, prostate-specificantigen, proteinase 3, prostein, Reg3a, RHAMM, ROPN1, SART2, SDCCAG8,SEL1L, SEPT1, SLC45A2, SPANX, SSX5, STXGALNAC1, STEAP4, survivin,TBC1D2, TEM1, TRP1, tumor antigens of epithelial origin, XAGE1, XAGE2,WT-1; overexpressed embryonic antigens such as CEA; overexpressedoncogenes and mutated tumor-suppressor genes such as p53, Ras,HER-2/neu; unique tumor antigens resulting from chromosomaltranslocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; andviral antigens, such as the Epstein Barr virus antigens EBVA and thehuman papillomavirus (HPV) antigens E6 and E7.

Other tumor antigens include, but are not limited to, TSP-180, MAGE-4,MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1,PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin,CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein,beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242,CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP,TPS, glioma-associated antigen, β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrinB2, CD19, CD20,CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

Typically, any anti-neoplastic agent that has activity versus asusceptible tumor being treated may be co-administered in the treatmentof cancer in the present invention. Examples of such agents can be foundin Cancer Principles and Practice of Oncology by V. T. Devita and S.Hellman (editors), 6^(th) edition (Feb. 15, 2001), Lippincott Williams &Wilkins Publishers. A person of ordinary skill in the art would be ableto discern which combinations of agents would be useful based on theparticular characteristics of the drugs and the cancer involved. Typicalanti-neoplastic agents useful in the present invention include, but arenot limited to, anti-microtubule agents such as diterpenoids and vincaalkaloids; platinum coordination complexes; alkylating agents such asnitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, andtriazenes; antibiotic agents such as anthracyclins, actinomycins andbleomycins; topoisomerase II inhibitors such as epipodophyllotoxins;antimetabolites such as purine and pyrimidine analogues and anti-folatecompounds; topoisomerase I inhibitors such as camptothecins; hormonesand hormonal analogues; signal transduction pathway inhibitors;non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeuticagents; proapoptotic agents; and cell cycle signalling inhibitors.

Examples of a further active ingredient or ingredients for use incombination or co-administered with the present ICOS binding protein areanti-neoplastic agents including any chemotherapeutic agents,immuno-modulatory agents or immune-modulators and immunostimulatoryadjuvants.

Anti-microtubule or anti-mitotic agents are phase specific agents activeagainst the microtubules of tumor cells during M or the mitosis phase ofthe cell cycle. Examples of anti-microtubule agents include, but are notlimited to, diterpenoids and vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specificanti-cancer agents that operate at the G₂/M phases of the cell cycle. Itis believed that the diterpenoids stabilize the β-tubulin subunit of themicrotubules, by binding with this protein. Disassembly of the proteinappears then to be inhibited with mitosis being arrested and cell deathfollowing. Examples of diterpenoids include, but are not limited to,paclitaxel and its analog docetaxel.

Paclitaxel, 5β,20-epoxy-1,2α,4,7β,10β,13α-hexa-hydroxytax-11-en-9-one4,10-diacetate 2-benzoate 13-ester with(2R,3S)—N-benzoyl-3-phenylisoserine; is a natural diterpene productisolated from the Pacific yew tree Taxus brevifolia and is commerciallyavailable as an injectable solution TAXOL®. It is a member of the taxanefamily of terpenes. It was first isolated in 1971 by Wani et al. J. Am.Chem, Soc., 93:2325. 1971), who characterized its structure by chemicaland X-ray crystallographic methods. One mechanism for its activityrelates to paclitaxel's capacity to bind tubulin, thereby inhibitingcancer cell growth. Schiff et al., Proc. Natl, Acad, Sci. USA,77:1561-1565 (1980); Schiff et al., Nature, 277:665-667 (1979); Kumar,J. Biol, Chem, 256: 10435-10441 (1981). For a review of synthesis andanticancer activity of some paclitaxel derivatives see: D. G. I.Kingston et al., Studies in Organic Chemistry vol. 26, entitled “Newtrends in Natural Products Chemistry 1986”, Attaur-Rahman, P. W. LeQuesne, Eds. (Elsevier, Amsterdam, 1986) pp 219-235.

Paclitaxel has been approved for clinical use in the treatment ofrefractory ovarian cancer in the United States (Markman et al., YaleJournal of Biology and Medicine, 64:583, 1991; McGuire et al., Ann.lntem, Med., 111:273, 1989) and for the treatment of breast cancer(Holmes et al., J. Nat. Cancer Inst., 83:1797, 1991.) It is a potentialcandidate for treatment of neoplasms in the skin (Einzig et. al., Proc.Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastireet. al., Sem. Oncol., 20:56, 1990). The compound also shows potentialfor the treatment of polycystic kidney disease (Woo et. al., Nature,368:750. 1994, lung cancer and malaria. Treatment of patients withpaclitaxel results in bone marrow suppression (multiple cell lineages,Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide, 1998) related tothe duration of dosing above a threshold concentration (50 nM) (Kearns,C. M. et. al., Seminars in Oncology, 3(6) p. 16-23, 1995).

Docetaxel, (2R,3S)—N-carboxy-3-phenylisoserine,N-tert-butyl ester,13-ester with 5β-20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate; is commercially available as aninjectable solution as TAXOTERE®. Docetaxel is indicated for thetreatment of breast cancer. Docetaxel is a semisynthetic derivative ofpaclitaxel q.v., prepared using a natural precursor,10-deacetyl-baccatin III, extracted from the needle of the European Yewtree. The dose limiting toxicity of docetaxel is neutropenia.

Vinca alkaloids are phase specific anti-neoplastic agents derived fromthe periwinkle plant. Vinca alkaloids act at the M phase (mitosis) ofthe cell cycle by binding specifically to tubulin. Consequently, thebound tubulin molecule is unable to polymerize into microtubules.Mitosis is believed to be arrested in metaphase with cell deathfollowing. Examples of vinca alkaloids include, but are not limited to,vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleukoblastine sulfate, is commercially available asVELBAN® as an injectable solution. Although, it has possible indicationas a second line therapy of various solid tumors, it is primarilyindicated in the treatment of testicular cancer and various lymphomasincluding Hodgkin's Disease; and lymphocytic and histiocytic lymphomas.Myelosuppression is the dose limiting side effect of vinblastine.

Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commerciallyavailable as ONCOVIN® as an injectable solution. Vincristine isindicated for the treatment of acute leukemias and has also found use intreatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.Alopecia and neurologic effects are the most common side effect ofvincristine and to a lesser extent myelosupression and gastrointestinalmucositis effects occur.

Vinorelbine, 3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine[R—(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)], commerciallyavailable as an injectable solution of vinorelbine tartrate(NAVELBINE®), is a semisynthetic vinca alkaloID Vinorelbine is indicatedas a single agent or in combination with other chemotherapeutic agents,such as cisplatin, in the treatment of various solid tumors,particularly non-small cell lung, advanced breast, and hormonerefractory prostate cancers. Myelosuppression is the most common doselimiting side effect of vinorelbine.

Platinum coordination complexes are non-phase specific anti-canceragents, which are interactive with DNA. The platinum complexes entertumor cells, undergo, aquation and form intra- and interstrandcrosslinks with DNA causing adverse biological effects to the tumor.Examples of platinum coordination complexes include, but are not limitedto, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available asPLATINOL® as an injectable solution. Cisplatin is primarily indicated inthe treatment of metastatic testicular and ovarian cancer and advancedbladder cancer. The primary dose limiting side effects of cisplatin arenephrotoxicity, which may be controlled by hydration and diuresis, andototoxicity.

Carboplatin, platinum, diammine[1,1-cyclobutane-dicarboxylate(2-)-O,O′], is commercially available asPARAPLATIN® as an injectable solution. Carboplatin is primarilyindicated in the first and second line treatment of advanced ovariancarcinoma. Bone marrow suppression is the dose limiting toxicity ofcarboplatin.

Alkylating agents are non-phase anti-cancer specific agents and strongelectrophiles. Typically, alkylating agents form covalent linkages, byalkylation, to DNA through nucleophilic moieties of the DNA moleculesuch as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazolegroups. Such alkylation disrupts nucleic acid function leading to celldeath. Examples of alkylating agents include, but are not limited to,nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil;alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; andtriazenes such as dacarbazine.

Cyclophosphamide,2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxidemonohydrate, is commercially available as an injectable solution ortablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent orin combination with other chemotherapeutic agents, in the treatment ofmalignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea,vomiting and leukopenia are the most common dose limiting side effectsof cyclophosphamide.

Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commerciallyavailable as an injectable solution or tablets as ALKERAN®. Melphalan isindicated for the palliative treatment of multiple myeloma andnon-resectable epithelial carcinoma of the ovary. Bone marrowsuppression is the most common dose limiting side effect of melphalan.

Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, iscommercially available as LEUKERAN® tablets. Chlorambucil is indicatedfor the palliative treatment of chronic lymphatic leukemia, andmalignant lymphomas such as lymphosarcoma, giant follicular lymphoma,and Hodgkin's disease. Bone marrow suppression is the most common doselimiting side effect of chlorambucil.

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially availableas MYLERAN® TABLETS. Busulfan is indicated for the palliative treatmentof chronic myelogenous leukemia. Bone marrow suppression is the mostcommon dose limiting side effects of busulfan.

Carmustine, 1,3-[bis(2-chloroethyl)-1-nitrosourea, is commerciallyavailable as single vials of lyophilized material as BiCNU®. Carmustineis indicated for the palliative treatment as a single agent or incombination with other agents for brain tumors, multiple myeloma,Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppressionis the most common dose limiting side effects of carmustine.

Dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, iscommercially available as single vials of material as DTIC-Dome®.Dacarbazine is indicated for the treatment of metastatic malignantmelanoma and in combination with other agents for the second linetreatment of Hodgkin's Disease. Nausea, vomiting, and anorexia are themost common dose limiting side effects of dacarbazine.

Antibiotic anti-neoplastics are non-phase specific agents, which bind orintercalate with DNA. Typically, such action results in stable DNAcomplexes or strand breakage, which disrupts ordinary function of thenucleic acids leading to cell death. Examples of antibioticanti-neoplastic agents include, but are not limited to, actinomycinssuch as dactinomycin, anthrocyclins such as daunorubicin anddoxorubicin; and bleomycins.

Dactinomycin, also know as Actinomycin D, is commercially available ininjectable form as COSMEGEN®. Dactinomycin is indicated for thetreatment of Wilm's tumor and rhabdomyosarcoma. Nausea, vomiting, andanorexia are the most common dose limiting side effects of dactinomycin.

Daunorubicin,(8S-cis+8-acetyl-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12naphthacenedione hydrochloride, is commercially available as a liposomalinjectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.Daunorubicin is indicated for remission induction in the treatment ofacute nonlymphocytic leukemia and advanced HIV associated Kaposi'ssarcoma. Myelosuppression is the most common dose limiting side effectof daunorubicin.

Doxorubicin, (8S,10S)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl,7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedionehydrochloride, is commercially available as an injectable form as RUBEX®or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatmentof acute lymphoblastic leukemia and acute myeloblastic leukemia, but isalso a useful component in the treatment of some solid tumors andlymphomas. Myelosuppression is the most common dose limiting side effectof doxorubicin.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated froma strain of Streptomyces verticillus, is commercially available asBLENOXANE®. Bleomycin is indicated as a palliative treatment, as asingle agent or in combination with other agents, of squamous cellcarcinoma, lymphomas, and testicular carcinomas. Pulmonary and cutaneoustoxicities are the most common dose limiting side effects of bleomycin.

Topoisomerase II inhibitors include, but are not limited to,epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derivedfrom the mandrake plant. Epipodophyllotoxins typically affect cells inthe S and G₂ phases of the cell cycle by forming a ternary complex withtopoisomerase II and DNA causing DNA strand breaks. The strand breaksaccumulate and cell death follows. Examples of epipodophyllotoxinsinclude, but are not limited to, etoposide and teniposide.

Etoposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-ethylidene-β-D-glucopyranoside], is commercially availableas an injectable solution or capsules as VePESID® and is commonly knownas VP-16. Etoposide is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of testicular andnon-small cell lung cancers. Myelosuppression is the most common sideeffect of etoposide. The incidence of leucopenia tends to be more severethan thrombocytopenia.

Teniposide, 4′-demethyl-epipodophyllotoxin9[4,6-0-(R)-thenylidene-β-D-glucopyranoside], is commercially availableas an injectable solution as VUMON® and is commonly known as VM-26.Teniposide is indicated as a single agent or in combination with otherchemotherapy agents in the treatment of acute leukemia in children.Myelosuppression is the most common dose limiting side effect ofteniposide. Teniposide can induce both leucopenia and thrombocytopenia.

Antimetabolite neoplastic agents are phase specific anti-neoplasticagents that act at S phase (DNA synthesis) of the cell cycle byinhibiting DNA synthesis or by inhibiting purine or pyrimidine basesynthesis and thereby limiting DNA synthesis. Consequently, S phase doesnot proceed and cell death follows. Examples of antimetaboliteanti-neoplastic agents include, but are not limited to, fluorouracil,methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4-(1H,3H) pyrimidinedione, is commerciallyavailable as fluorouracil. Administration of 5-fluorouracil leads toinhibition of thymidylate synthesis and is also incorporated into bothRNA and DNA. The result typically is cell death. 5-fluorouracil isindicated as a single agent or in combination with other chemotherapyagents in the treatment of carcinomas of the breast, colon, rectum,stomach and pancreas. Myelosuppression and mucositis are dose limitingside effects of 5-fluorouracil. Other fluoropyrimidine analogs include5-fluoro deoxyuridine (floxuridine) and 5-fluorodeoxyuridinemonophosphate.

Cytarabine, 4-amino-1-β-D-arabinofuranosyl-2 (1H)-pyrimidinone, iscommercially available as CYTOSAR-U® and is commonly known as Ara-C. Itis believed that cytarabine exhibits cell phase specificity at S-phaseby inhibiting DNA chain elongation by terminal incorporation ofcytarabine into the growing DNA chain. Cytarabine is indicated as asingle agent or in combination with other chemotherapy agents in thetreatment of acute leukemia. Other cytidine analogs include5-azacytidine and 2′,2′-difluorodeoxycytidine (gemcitabine). Cytarabineinduces leucopenia, thrombocytopenia, and mucositis.

Mercaptopurine, 1,7-dihydro-6H-purine-6-thione monohydrate, iscommercially available as PURINETHOL®. Mercaptopurine exhibits cellphase specificity at S-phase by inhibiting DNA synthesis by an as of yetunspecified mechanism. Mercaptopurine is indicated as a single agent orin combination with other chemotherapy agents in the treatment of acuteleukemia. Myelosuppression and gastrointestinal mucositis are expectedside effects of mercaptopurine at high doses. A useful mercaptopurineanalog is azathioprine.

Thioguanine, 2-amino-1,7-dihydro-6H-purine-6-thione, is commerciallyavailable as TABLOID®. Thioguanine exhibits cell phase specificity atS-phase by inhibiting DNA synthesis by an as of yet unspecifiedmechanism. Thioguanine is indicated as a single agent or in combinationwith other chemotherapy agents in the treatment of acute leukemia.Myelosuppression, including leucopenia, thrombocytopenia, and anemia, isthe most common dose limiting side effect of thioguanine administration.However, gastrointestinal side effects occur and can be dose limiting.Other purine analogs include pentostatin, erythrohydroxynonyladenine,fludarabine phosphate, and cladribine.

Gemcitabine, 2′-deoxy-2′, 2′-difluorocytidine monohydrochloride(β-isomer), is commercially available as GEMZAR®. Gemcitabine exhibitscell phase specificity at S-phase and by blocking progression of cellsthrough the G1/S boundary. Gemcitabine is indicated in combination withcisplatin in the treatment of locally advanced non-small cell lungcancer and alone in the treatment of locally advanced pancreatic cancer.Myelosuppression, including leucopenia, thrombocytopenia, and anemia, isthe most common dose limiting side effect of gemcitabine administration.

Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid, is commercially availableas methotrexate sodium. Methotrexate exhibits cell phase effectsspecifically at S-phase by inhibiting DNA synthesis, repair and/orreplication through the inhibition of dyhydrofolic acid reductase whichis required for synthesis of purine nucleotides and thymidylate.Methotrexate is indicated as a single agent or in combination with otherchemotherapy agents in the treatment of choriocarcinoma, meningealleukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head,neck, ovary and bladder. Myelosuppression (leucopenia, thrombocytopenia,and anemia) and mucositis are expected side effect of methotrexateadministration.

Camptothecins, including, camptothecin and camptothecin derivatives areavailable or under development as Topoisomerase I inhibitors.Camptothecins cytotoxic activity is believed to be related to itsTopoisomerase I inhibitory activity. Examples of camptothecins include,but are not limited to irinotecan, topotecan, and the various opticalforms of7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20-camptothecindescribed below.

Irinotecan HCl, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dionehydrochloride, is commercially available as the injectable solutionCAMPTOSAR®.

Irinotecan is a derivative of camptothecin which binds, along with itsactive metabolite SN-38, to the topoisomerase I-DNA complex. It isbelieved that cytotoxicity occurs as a result of irreparable doublestrand breaks caused by interaction of the topoisomerase I:DNA:irintecanor SN-38 ternary complex with replication enzymes. Irinotecan isindicated for treatment of metastatic cancer of the colon or rectum. Thedose limiting side effects of irinotecan HCl are myelosuppression,including neutropenia, and GI effects, including diarrhea.

Topotecan HCl,(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3′,4′,6,7]indolizino[1,2-b]quinoline-3,14-(4H,12H)-dionemonohydrochloride, is commercially available as the injectable solutionHYCAMTIN®. Topotecan is a derivative of camptothecin which binds to thetopoisomerase I-DNA complex and prevents religation of singles strandbreaks caused by Topoisomerase I in response to torsional strain of theDNA molecule. Topotecan is indicated for second line treatment ofmetastatic carcinoma of the ovary and small cell lung cancer. The doselimiting side effect of topotecan HCl is myelosuppression, primarilyneutropenia.

Rituximab is a chimeric monoclonal antibody which is sold as RITUXAN®and MABTHERA®. Rituximab binds to CD20 on B cells and causes cellapoptosis. Rituximab is administered intravenously and is approved fortreatment of rheumatoid arthritis and B-cell non-Hodgkin's lymphoma.

Ofatumumab is a fully human monoclonal antibody which is sold asARZERRA®. Ofatumumab binds to CD20 on B cells and is used to treatchronic lymphocytic leukemia CLL; a type of cancer of the white bloodcells) in adults who are refractory to treatment with fludarabine(Fludara) and alemtuzumab Campath).

Trastuzumab (HEREPTIN®) is a humanized monoclonal antibody that binds tothe HER2 receptor. It original indication is HER2 positive breastcancer.

Cetuximab (ERBITUX®) is a chimeric mouse human antibody that inhibitsepidermal growth factor receptor (EGFR).

mTOR inhibitors include but are not limited to rapamycin (FK506) andrapalogs, RAD001 or everolimus (Afinitor), CCI-779 or temsirolimus,AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and Pp121.

Bexarotene is sold as Targretin® and is a member of a subclass ofretinoids that selectively activate retinoid X receptors (RXRs). Theseretinoid receptors have biologic activity distinct from that of retinoicacid receptors (RARs). The chemical name is4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl]benzoic acID Bexarotene is used to treat cutaneous T-celllymphoma CTCL, a type of skin cancer) in people whose disease could notbe treated successfully with at least one other medication.

Sorafenib marketed as Nexavar® is in a class of medications calledmultikinase inhibitors. Its chemical name is4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide.Sorafenib is used to treat advanced renal cell carcinoma (a type ofcancer that begins in the kidneys). Sorafenib is also used to treatunresectable hepatocellular carcinoma (a type of liver cancer thatcannot be treated with surgery).

Examples of erbB inhibitors include lapatinib, erlotinib, and gefitinib.Lapatinib,N-(3-chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)-6-[5-({[2-(methylsulfonyl)ethyl]amino}methyl)-2-furanyl]-4-quinazolinamine(represented by formula II, as illustrated), is a potent, oral,small-molecule, dual inhibitor of erbB-1 and erbB-2 (EGFR and HER2)tyrosine kinases that is approved in combination with capecitabine forthe treatment of HER2-positive metastatic breast cancer.

The free base, HCl salts, and ditosylate salts of the compound offormula (II) may be prepared according to the procedures disclosed in WO99/35146, published Jul. 15, 1999; and WO 02/02552 published Jan. 10,2002.

Erlotinib,N-(3-ethynylphenyl)-6,7-bis{[2-(methyloxy)ethyl]oxy}-4-quinazolinamineCommercially available under the tradename Tarceva) is represented byformula III, as illustrated:

The free base and HCl salt of erlotinib may be prepared, for example,according to U.S. Pat. No. 5,747,498, Example 20.

Gefitinib,4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4-morpholin)propoxy]is represented by formula IV, as illustrated:

Gefitinib, which is commercially available under the trade name IRESSA®(Astra-Zenenca) is an erbB-1 inhibitor that is indicated as monotherapyfor the treatment of patients with locally advanced or metastaticnon-small-cell lung cancer after failure of both platinum-based anddocetaxel chemotherapies. The free base, HCl salts, and diHCl salts ofgefitinib may be prepared according to the procedures of InternationalPatent Application No. PCT/GB96/00961, filed Apr. 23, 1996, andpublished as WO 96/33980 on Oct. 31, 1996.

Also of interest, is the camptothecin derivative of formula A following,currently under development, including the racemic mixture (R,S) form aswell as the R and S enantiomers:

known by the chemical name“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R,S)-camptothecin(racemic mixture) or“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(R)-camptothecin(R enantiomer) or“7-(4-methylpiperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin(S enantiomer). Such compound as well as related compounds aredescribed, including methods of making, in U.S. Pat. Nos. 6,063,923;5,342,947; 5,559,235; 5,491,237 and pending U.S. patent application Ser.No. 08/977,217 filed Nov. 24, 1997.

Hormones and hormonal analogues are useful compounds for treatingcancers in which there is a relationship between the hormone(s) andgrowth and/or lack of growth of the cancer. Examples of hormones andhormonal analogues useful in cancer treatment include, but are notlimited to, adrenocorticosteroids such as prednisone and prednisolonewhich are useful in the treatment of malignant lymphoma and acuteleukemia in children; aminoglutethimide and other aromatase inhibitorssuch as anastrozole, letrazole, vorazole, and exemestane useful in thetreatment of adrenocortical carcinoma and hormone dependent breastcarcinoma containing estrogen receptors; progestrins such as megestrolacetate useful in the treatment of hormone dependent breast cancer andendometrial carcinoma; estrogens, androgens, and anti-androgens such asflutamide, nilutamide, bicalutamide, cyproterone acetate and5α-reductases such as finasteride and dutasteride, useful in thetreatment of prostatic carcinoma and benign prostatic hypertrophy;anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene,iodoxyfene, as well as selective estrogen receptor modulators (SERMS)such those described in U.S. Pat. Nos. 5,681,835, 5,877,219, and6,207,716, useful in the treatment of hormone dependent breast carcinomaand other susceptible cancers; and gonadotropin-releasing hormone (GnRH)and analogues thereof which stimulate the release of leutinizing hormone(LH) and/or follicle stimulating hormone (FSH) for the treatmentprostatic carcinoma, for instance, LHRH agonists and antagagonists suchas goserelin acetate and luprolide.

Signal transduction pathway inhibitors are those inhibitors, which blockor inhibit a chemical process which evokes an intracellular change. Asused herein this change is cell proliferation or differentiation. Signaltransduction inhibitors useful in the present invention includeinhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases,SH2/SH3domain blockers, serine/threonine kinases, phosphotidylinositol-3 kinases, myo-inositol signalling, and Ras oncogenes.

Several protein tyrosine kinases catalyse the phosphorylation ofspecific tyrosyl residues in various proteins involved in the regulationof cell growth. Such protein tyrosine kinases can be broadly classifiedas receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having anextracellular ligand binding domain, a transmembrane domain, and atyrosine kinase domain. Receptor tyrosine kinases are involved in theregulation of cell growth and are generally termed growth factorreceptors. Inappropriate or uncontrolled activation of many of thesekinases, i.e. aberrant kinase growth factor receptor activity, forexample by over-expression or mutation, has been shown to result inuncontrolled cell growth. Accordingly, the aberrant activity of suchkinases has been linked to malignant tissue growth. Consequently,inhibitors of such kinases could provide cancer treatment methods.Growth factor receptors include, for example, epidermal growth factorreceptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2,erbB4, vascular endothelial growth factor receptor (VEGFr), tyrosinekinase with immunoglobulin-like and epidermal growth factor homologydomains (TIE-2), insulin growth factor-I (IGFI) receptor, macrophagecolony stimulating factor Cfms), BTK, ckit, cmet, fibroblast growthfactor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin(eph) receptors, and the RET protooncogene. Several inhibitors of growthreceptors are under development and include ligand antagonists,antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.Growth factor receptors and agents that inhibit growth factor receptorfunction are described, for instance, in Kath, John C., Exp. Opin. Ther.Patents (2000) 10(6):803-818; Shawver et al DDT Vol 2, No. 2 Feb. 1997;and Lofts, F. J. et al, “Growth factor receptors as targets”, NewMolecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr,David, CRC press 1994, London.

Tyrosine kinases, which are not growth factor receptor kinases aretermed non-receptor tyrosine kinases. Non-receptor tyrosine kinasesuseful in the present invention, which are targets or potential targetsof anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focaladhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Suchnon-receptor kinases and agents which inhibit non-receptor tyrosinekinase function are described in Sinh, S. and Corey, S. J., (1999)Journal of Hematotherapy and Stem Cell Research 8 (5): 465-80; andBolen, J. B., Brugge, J. S., (1997) Annual review of Immunology. 15:371-404.

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domainbinding in a variety of enzymes or adaptor proteins including, PI3-K p85subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) andRas-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussedin Smithgall, T. E. (1995), Journal of Pharmacological and ToxicologicalMethods. 34 (3) 125-32.

Inhibitors of Serine/Threonine Kinases including MAP kinase cascadeblockers which include blockers of Raf kinases (rafk), Mitogen orExtracellular Regulated Kinase (MEKs), and Extracellular RegulatedKinases (ERKs); and Protein kinase C family member blockers includingblockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta).IkB kinase family (IKKa, IKKb), PKB family kinases, AKT kinase familymembers, and TGF beta receptor kinases. Such Serine/Threonine kinasesand inhibitors thereof are described in Yamamoto, T., Taya, S.,Kaibuchi, K., (1999), Journal of Biochemistry. 126 (5) 799-803; Brodt,P, Samani, A., and Navab, R. (2000), Biochemical Pharmacology, 60.1101-1107; Massague, J., Weis-Garcia, F. (1996) Cancer Surveys.27:41-64; Philip, P. A., and Harris, A. L. (1995), Cancer Treatment andResearch. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal ChemistryLetters, (10), 2000, 223-226; U.S. Pat. No. 6,268,391; andMartinez-Iacaci, L., et al, Int. J. Cancer (2000), 88(1), 44-52.

Inhibitors of Phosphotidyl inositol-3 Kinase family members includingblockers of PI3-kinase, ATM, DNA-PK, and Ku are also useful in thepresent invention. Such kinases are discussed in Abraham, R. T. (1996),Current Opinion in Immunology. 8 (3) 412-8; Canman, C. E., Lim, D. S.(1998), Oncogene 17 (25) 3301-3308; Jackson, S. P. (1997), InternationalJournal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H. etal, Cancer res, (2000) 60(6), 1541-1545.

Also useful in the present invention are Myo-inositol signallinginhibitors such as phospholipase C blockers and Myoinositol analogues.Such signal inhibitors are described in Powis, G., and Kozikowski A.,(1994 New Molecular Targets for Cancer Chemotherapy ed., Paul Workmanand David Kerr, CRC press 1994, London.

Another group of signal transduction pathway inhibitors are inhibitorsof Ras Oncogene. Such inhibitors include inhibitors offarnesyltransferase, geranyl-geranyl transferase, and CAAX proteases aswell as anti-sense oligonucleotides, ribozymes and immunotherapy. Suchinhibitors have been shown to block ras activation in cells containingwild type mutant ras, thereby acting as antiproliferation agents. Rasoncogene inhibition is discussed in Scharovsky, O. G., Rozados, V. R.,Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7 (4292-8; Ashby, M. N. (1998), Current Opinion in Lipidology. 9 (2) 99-102;and Bennett, C. F. and Cowsert, L. M. BioChim. Biophys. Acta, (1999)1489(1):19-30.

As mentioned above, antibody antagonists to receptor kinase ligandbinding may also serve as signal transduction inhibitors. This group ofsignal transduction pathway inhibitors includes the use of humanizedantibodies to the extracellular ligand binding domain of receptortyrosine kinases. For example Imclone C225 EGFR specific antibody (seeGreen, M. C. et al, Monoclonal Antibody Therapy for Solid Tumors, CancerTreat. Rev., (2000), 26 (4, 269-286); Herceptin® erbB2 antibody (seeTyrosine Kinase Signalling in Breast cancer:erbB Family ReceptorTyrosine Kniases, Breast cancer Res., 2000, 2(3), 176-183); and 2CBVEGFR2 specific antibody (see Brekken, R. A. et al, Selective Inhibitionof VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumorgrowth in mice, Cancer Res. (2000) 60, 5117-5124.

Non-receptor kinase angiogenesis inhibitors may also find use in thepresent invention. Inhibitors of angiogenesis related VEGFR and TIE2 arediscussed above in regard to signal transduction inhibitors (bothreceptors are receptor tyrosine kinases). Angiogenesis in general islinked to erbB2/EGFR signaling since inhibitors of erbB2 and EGFR havebeen shown to inhibit angiogenesis, primarily VEGF expression. Thus, thecombination of an erbB2/EGFR inhibitor with an inhibitor of angiogenesismakes sense. Accordingly, non-receptor tyrosine kinase inhibitors may beused in combination with the EGFR/erbB2 inhibitors of the presentinvention. For example, anti-VEGF antibodies, which do not recognizeVEGFR (the receptor tyrosine kinase), but bind to the ligand; smallmolecule inhibitors of integrin (alpha, beta₃) that will inhibitangiogenesis; endostatin and angiostatin (non-RTK) may also prove usefulin combination with the disclosed erb family inhibitors. (See Bruns C Jet al (2000), Cancer Res., 60: 2926-2935; Schreiber A B, Winkler M E,and Derynck R. (1986), Science, 232: 1250-1253; Yen L et al. (2000),Oncogene 19: 3460-3469).

Agents used in immunotherapeutic regimens may also be useful incombination with the compounds of formula (I). There are a number ofimmunologic strategies to generate an immune response against erbB2 orEGFR. These strategies are generally in the realm of tumor vaccinations.The efficacy of immunologic approaches may be greatly enhanced throughcombined inhibition of erbB2/EGFR signaling pathways using a smallmolecule inhibitor. Discussion of the immunologic/tumor vaccine approachagainst erbB2/EGFR are found in Reilly R T et al. (2000), Cancer Res.60: 3569-3576; and Chen Y, Hu D, Eling D J, Robbins J, and Kipps T J.(1998), Cancer Res. 58: 1965-1971.

Agents used in proapoptotic regimens (e.g., bcl-2 antisenseoligonucleotides) may also be used in the combination of the presentinvention. Members of the Bcl-2 family of proteins block apoptosis.Upregulation of bcl-2 has therefore been linked to chemoresistance.Studies have shown that the epidermal growth factor (EGF) stimulatesanti-apoptotic members of the bcl-2 family (i.e., mcl-1). Therefore,strategies designed to downregulate the expression of bcl-2 in tumorshave demonstrated clinical benefit and are now in Phase II/III trials,namely Genta's G3139 bcl-2 antisense oligonucleotide. Such proapoptoticstrategies using the antisense oligonucleotide strategy for bcl-2 arediscussed in Water J S et al. (2000), J. Clin. Oncol. 18: 1812-1823; andKitada S et al. (1994, Antisense Res. Dev. 4: 71-79.

Trastuzumab (HEREPTIN®) is a humanized monoclonal antibody that binds tothe HER2 receptor. It original indication is HER2 positive breastcancer.

Trastuzumab emtansine (trade name Kadcyla) is anantibody-drug conjugateconsisting of the monoclonal antibody trastuzumab (Herceptin) linked tothe cytotoxic agent mertansine (DM1). Trastuzumab alone stops growth ofcancer cells by binding to the HER2/neu receptor, whereas mertansineenters cells and destroys them by binding to tubulin. Because themonoclonal antibody targets HER2, and HER2 is only overexpressed incancer cells, the conjugate delivers the toxin specifically to tumorcells. The conjugate is abbreviated T-DM1.

Cetuximab (ERBITUX®) is a chimeric mouse human antibody that inhibitsepidermal growth factor receptor (EGFR).

Pertuzumab (also called 2C4, trade name Omnitarg) is a monoclonalantibody. The first of its class in a line of agents called “HERdimerization inhibitors”. By binding to HER2, it inhibits thedimerization of HER2 with other HER receptors, which is hypothesized toresult in slowed tumor growth. Pertuzumab is described in WO01/00245published Jan. 4, 2001.

Rituximab is a chimeric monoclonal antibody which is sold as RITUXAN®and MABTHERA®. Rituximab binds to CD20 on B cells and causes cellapoptosis. Rituximab is administered intravenously and is approved fortreatment of rheumatoid arthritis and B-cell non-Hodgkin's lymphoma.

Ofatumumab is a fully human monoclonal antibody which is sold asARZERRA®. Ofatumumab binds to CD20 on B cells and is used to treatchronic lymphocytic leukemia (CLL; a type of cancer of the white bloodcells) in adults who are refractory to treatment with fludarabine(Fludara) and alemtuzumab (Campath).

Cell cycle signalling inhibitors inhibit molecules involved in thecontrol of the cell cycle. A family of protein kinases called cyclindependent kinases CDKs) and their interaction with a family of proteinstermed cyclins controls progression through the eukaryotic cell cycle.The coordinate activation and inactivation of different cyclin/CDKcomplexes is necessary for normal progression through the cell cycle.Several inhibitors of cell cycle signalling are under development. Forinstance, examples of cyclin dependent kinases, including CDK2, CDK4,and CDK6 and inhibitors for the same are described in, for instance,Rosania et al, Exp. Opin. Ther. Patents (2000) 10(2):215-230.

As used herein “immuno-modulators” refer to any substance includingmonoclonal antibodies that effects the immune system. The ICOS bindingproteins of the present invention can be considered immune-modulators.Immuno-modulators can be used as anti-neoplastic agents for thetreatment of cancer. For example, immune-modulators include, but are notlimited to, anti-CTLA-4 antibodies such as ipilimumab (YERVOY) andanti-PD-1 antibodies (Opdivo/nivolumab and Keytruda/pembrolizumab).Other immuno-modulators include, but are not limited to, OX-40antibodies, PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, 41BBantibodies and GITR antibodies.

Yervoy (ipilimumab) is a fully human CTLA-4 antibody marketed by BristolMyers Squibb. The protein structure of ipilimumab and methods are usingare described in U.S. Pat. Nos. 6,984,720 and 7,605,238.

Opdivo/nivolumab is a fully human monoclonal antibody marketed byBristol Myers Squibb directed against the negative immunoregulatoryhuman cell surface receptor PD-1 (programmed death-1 or programmed celldeath-1/PCD-1) with immunopotentiation activity. Nivolumab binds to andblocks the activation of PD-1, an Ig superfamily transmembrane protein,by its ligands PD-L1 and PD-L2; resulting in the activation of T-cellsand cell-mediated immune responses against tumor cells or pathogens.Activated PD-1 negatively regulates T-cell activation and effectorfunction through the suppression of P13k/Akt pathway activation. Othernames for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. Theamino acid sequence for nivolumab and methods of using and making aredisclosed in U.S. Pat. No. 8,008,449.

KEYTRUDA/pembrolizumab is an anti-PD-1 antibodies marketed for thetreatment of lung cancer by Merck. The amino acid sequence ofpembrolizumab and methods of using are disclosed in U.S. Pat. No.8,168,757.

CD134, also known as OX40, is a member of the TNFR-superfamily ofreceptors which is not constitutively expressed on resting naïve Tcells, unlike CD28. OX40 is a secondary costimulatory molecule,expressed after 24 to 72 hours following activation; its ligand, OX40L,is also not expressed on resting antigen presenting cells, but isfollowing their activation. Expression of OX40 is dependent on fullactivation of the T cell; without CD28, expression of OX40 is delayedand of fourfold lower levels. OX-40 antibodies, OX-40 fusion proteinsand methods of using them are disclosed in: U.S. Pat. No. 7,504,101;U.S. Pat. No. 7,758,852; U.S. Pat. No. 7,858,765; U.S. Pat. No.7,550,140; U.S. Pat. No. 7,960,515; WO2012027328; WO2013028231.

Antibodies to PD-L1 (also referred to as CD274 or B7-H1) and methods foruse are disclosed in U.S. Pat. No. 7,943,743; U.S. Pat. No. 8,383,796;US20130034559, WO2014055897, U.S. Pat. No. 8,168,179; and U.S. Pat. No.7,595,048. PD-L1 antibodies are in development as immuno-modulatoryagents for the treatment of cancer.

As used herein “immunostimulatory agent” refers to any agent that canstimulate the immune system. As used herein immunostimulatory agentsinclude, but are not limited to, vaccine adjuvants.

Aminoalkyl glucosaminide phosphates (AGPs) are known to be useful asvaccine adjuvants and immunostimulatory agents for stimulating cytokineproduction, activating macrophages, promoting innate immune response,and augmenting antibody production in immunized animals. Aminoalkylglucosaminide phosphates (AGPs) are synthetic ligands of the Toll-likeReceptor 4 (TLR4). AGPs and their immunomodulating effects via TLR4 aredisclosed in patent publications such as WO 2006/016997, WO 2001/090129,and/or U.S. Pat. No. 6,113,918 and have been reported in the literature.Additional AGP derivatives are disclosed in U.S. Pat. No. 7,129,219,U.S. Pat. No. 6,525,028 and U.S. Pat. No. 6,911,434. Certain AGPs act asagonists of TLR4, while others are recognized as TLR4 antagonists.

Aminoalkyl glucosaminide phosphate compounds employed in the presentinvention have the structure set forth in Formula 1 as follows:

wherein

-   -   m is 0 to 6    -   n is 0 to 4;    -   X is O or S, preferably O;    -   Y is O or NH;    -   Z is O or H;    -   each R₁, R₂, R₃ is selected independently from the group        consisting of a C₁₋₂₀ acyl and a C₁₋₂₀ alkyl;    -   R₄ 1 S H or Me;    -   R₅ is selected independently from the group consisting of —H,        —OH, —(C₁-C₄) alkoxy, —PO₃R₈R₉, —OPO₃R₈R₉, —SO₃R₈, —OSO₃R₈,        —NR₈R₉, —SR₈, —CN, —NO₂, —CHO, —CO₂R₈, and —CONR₈R₉, wherein R₈        and R₉ are each independently selected from H and (C₁-C₄) alkyl;        and    -   each R₆ and R₇ is independently H or PO₃H₂.

In Formula 1 the configuration of the 3′ stereogenic centers to whichthe normal fatty acyl residues (that is, the secondary acyloxy or alkoxyresidues, e.g., R₁O, R₂O, and R₃O) are attached is R or S, preferably R(as designated by Cahn-Ingold-Prelog priority rules). Configuration ofaglycon stereogenic centers to which R₄ and R₅ are attached can be R orS. All stereoisomers, both enantiomers and diastereomers, and mixturesthereof, are considered to fall within the scope of the presentinvention.

The number of carbon atoms between heteroatom X and the aglycon nitrogenatom is determined by the variable “n”, which can be an integer from 0to 4, preferably an integer from 0 to 2.

The chain length of normal fatty acids R₁, R₂, and R₃ can be from about6 to about 16 carbons, preferably from about 9 to about 14 carbons. Thechain lengths can be the same or different. Some preferred embodimentsinclude chain lengths where R1, R2 and R3 are 6 or 10 or 12 or 14.

Formula 1 encompasses L/D-seryl, -threonyl, -cysteinyl ether and esterlipid AGPs, both agonists and antagonists and their homologs (n=1-4), aswell as various carboxylic acid bioisosteres (i.e, R₅ is an acidic groupcapable of salt formation; the phosphate can be either on 4- or6-position of the glucosamine unit, but preferably is in the4-position).

In a preferred embodiment of the invention employing an AGP compound ofFormula 1, n is 0, R₅ is CO₂H, R₆ is PO₃H₂, and R₇ is H. This preferredAGP compound is set forth as the structure in Formula 1a as follows:

wherein X is O or S; Y is O or NH; Z is O or H; each R₁, R₂, R₃ isselected independently from the group consisting of a C₁₋₂₀ acyl and aC₁₋₂₀ alkyl; and R₄ is H or methyl.

In Formula 1a the configuration of the 3′ stereogenic centers to whichthe normal fatty acyl residues (that is, the secondary acyloxy or alkoxyresidues, e.g., R₁O, R₂O, and R₃O) are attached as R or S, preferably R(as designated by Cahn-Ingold-Prelog priority rules). Configuration ofaglycon stereogenic centers to which R₄ and CO₂H are attached can be Ror S. All stereoisomers, both enantiomers and diastereomers, andmixtures thereof, are considered to fall within the scope of the presentinvention.

Formula 1a encompasses L/D-seryl, -threonyl, -cysteinyl ether or esterlipid AGPs, both agonists and antagonists.

In both Formula 1 and Formula 1a, Z is O attached by a double bond ortwo hydrogen atoms which are each attached by a single bond. That is,the compound is ester-linked when Z═Y═O; amide-linked when Z═O and Y═NH;and ether-linked when Z═H/H and Y═O.

Especially preferred compounds of Formula 1 are referred to as CRX-601and CRX-527. Their structures are set forth as follows:

Additionally, another preferred embodiment employs CRX 547 having thestructure shown.

Still other embodiments include AGPs such as CRX 602 or CRX 526providing increased stability to AGPs having shorter secondary acyl oralkyl chains.

In one embodiment methods are provided for treating cancer in a mammalin need thereof, which comprises: administering to such mammal atherapeutically effective amount of:

-   -   an ICOS binding protein of the present invention,    -   and b) at least one anti-neoplastic agent.

In one embodiment methods are provided for treating cancer in a mammalin need thereof, which comprises: administering to such mammal atherapeutically effective amount of:

-   -   an ICOS binding protein of the present invention,    -   and b) at least one second immuno-modulatory agent.

In one embodiment said second immune-modulatory agent is selected fromthe group of: an anti-CTLA4 antibody, an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-OX40 antibody, an anti-GITR antibody, andanti-41BB antibody, an anti-LAG3 antibody and an anti-TIM3 antibody.

In one embodiment methods are provided for treating cancer in a mammalin need thereof, which comprises: administering to such mammal atherapeutically effective amount of:

-   -   an ICOS binding protein or the present invention,    -   and b) at least one immuno-stimulatory agent.

In one embodiment, the immune-stimulatory agent is a TLR4 agonist. Inone embodiment the immune-stimulatory agent is an AGP. In one aspect,the immune-stimulatory agent is a compound of Formula I. In one aspect,it is a compound of Formula 1a. In one aspect, the immune-stimulatoryagent is selected from the group consisting of: CRX-601, CRX-547,CRX-602, CRX-527, and CRX-526.

EXAMPLES

The following examples illustrate various non-limiting aspects of thisinvention.

Example 1 ICOS Expression in Cancer

In general, solid tumors appear to have low levels of infiltrating ICOS⁺T cells, consistent with the theory that ICOS mediates anti-tumor immuneresponses. ICOS expression analyses across various tumor histologies wasgenerated using publically available mRNA expression datasets from TheCancer Genome Atlas (TCGA) and other databases. Table 3 shows therelative percentage of tumors from each histology that showed somedetectable level of ICOS mRNA expression. This analysis identifies tumorhistologies known to be sensitive to other cancer immunotherapyapproaches (melanoma, RCC, NSCLC) as having a relatively higherpercentage of tumors (>10%) which have detectable ICOS⁺ tumorinfiltrating lymphocytes (TILs), whereas tumor types known to be poorlyimmunogenic (prostate, ovarian, and pancreatic) having relatively lowerpercentage of tumors which have ICOS⁺ TILs (<10%) (Table 3).Interestingly, tumor types known to be associated with viral infectionand/or chronic inflammation (H&N, Gastric, Esophageal, and Cervical)were among the tumor types showing the highest percentage of ICOS⁺ TILs.What is not clear from these mRNA expression analyses is thesubpopulation of TILs which express ICOS in each respective tumor type.In some instances ICOS may be predominantly expressed on tumorinfiltrating T_(regs), while in others it may indicate level of ICOS⁺ Teffector cell infiltration.

TABLE 3 ICOS mRNA expression across different tumor types Tumor TypeTotal N ICOS+ (N) ICOS+ (Per.) H&N 426 157 36.9% Gastric 285 75 26.3%Esophageal 70 17 24.3% Melanoma (M) 295 69 23.4% NSCLC(AD) 501 112 22.4%NSCLC(SCC) 489 85 17.4% Cervical 185 32 17.3% Breast 1048 162 15.5%Bladder 244 35 14.3% RCC 522 64 12.3% Melanoma (P) 82 7 8.5% Pancreas 857 8.2% Colon 446 34 7.6% Thyroid 498 34 6.8% HCC 191 11 5.8% Sarcoma 1034 3.9% Ovarian 412 13 3.2% Prostate 336 10 3.0% Endometrial 532 15 2.8%Rectal 163 4 2.5% GBM 156 0 0.0%

Analysis of ICOS expression by immunohistochemistry (IHC) in primaryhuman non-small cell lung carcinoma (NSCLC), triple-negative breastcancer (TNBC), colo-rectal cancer (CRC), prostate, pancreatic, ovarian,renal cell cancer (RCC) and melanoma was performed to better understandwhich subsets of TILs are associated with ICOS expression in differenttumor types (Table 4). Similar to what was observed in the mRNAexpression analysis, the abundance of ICOS⁺ TILs were relatively low,even in individual tumors where large numbers of CD4⁺, CD8⁺ and/orFoxP3⁺ TILs were otherwise present. Again, melanoma, renal cellcarcinoma (RCC) and non-small cell lung carcinoma (NSCLC) histologieshad the highest percentage of tumors with some level of detectable ICOS⁺infiltrate (Table 4, Column 2). Conversely, prostate, ovarian andpancreatic tumors showed almost no ICOS⁺ TILs (Table 4). These analysesclearly show that solid tumors have low basal levels of ICOS⁺ TILs andmay benefit from the expansion and functional induction of thispopulation of cells. Future studies using flow cytometry and dual-colorimmunohistochemistry to analyze primary human tumors will help determinewhich specific T cells subsets express ICOS.

TABLE 4 Average number of positive cells (range) Entity No. of samplesICOS ICOS CD3 CD4 CD8 FOXp3 NSCLC n = 17 − 0 11 (0-74) 2 (0-9) 20(0-147) 10 (0-28) (squamous) n = 23 + 3 (0-25) 36 (2-143) 8 (0-26) 39(5-188) 18 (0-75) NSCLC n = 15 − 0 36 (0-157) 4 (0-20) 71 (5-238) 10(1-41) (adenocarcinoma) n = 25 + 2 (0-7)  56 (0-181) 8 (0-39) 69(10-201) 19 (0-55) TNBC n = 24 − 0 14 (0-91) 9 (0-132) 17 (0-95) 6(0-25) n = 11 + 5 (0-20) 85 (3-259) 13 (0-45) 113 (2-393) 30 (2-81) CRCn = 22 − 0 12 (0-47) 14 (0-44) 20 (0-83) 14 (2-52) n = 23 + 2 (0-13) 31(5-101) 22 (5-48) 53 (4-151) 24 (5-43) Prostate Cancer n = 29 − 0 10(0-78) 17 (1-96) 23 (1-176) 5 (0-25) n = 1 + 1 30 48 55 11 PancreaticCancer n = 11 − 0 15 (1-32) 17 (3-71) 20 (3-81) 6 (0-21) n = 4 + 0(0-1)  31 (7-85) 17 (6-31) 17 (2-51) 4 (0-9) Ovarian Cancer n = 15 − 013 (0-105) 14 (1-78) 13 (0-83) 2 (0-12) n = 5 + 1 (0-3)  19 (6-35) 13(6-19) 32 (10-76) 7 (4-13) RCC n = 3 − 0 50 (14-104) 58 (36-85) 56(24-119) 4 (0-9) n = 7 + 5 (1-16) 45 (10-130) 85 (26-164) 71 (12-232) 15(3-28) Melanoma n = 7 − 0 42 (1-155) 12 (1-31) 35 (0-156) 5 (0-10) n =12 + 7 (0-21) 84 (13-222) 32 (13-70) 69 (26-179) 19 (6-40)

Example 2 Screening of ICOS Antibody Agonist Isolation of Primary HumanPBMC

Fresh blood was obtained from blood donors and was diluted 1:1 withPhenol red free-10% RPMI1640 media. Diluted blood was layered on top ofthe density medium in a Uni-Sep Max 50 ml conical tube and centrifuge at400×g for 20 minutes at room temperature with BREAK OFF. The resultedwhite mononuclear layer (buffy coat) was carefully extracted into a new50 mL conical tube through a 100 μM cell strainer. An equal volume ofPhenol red free-10% RPMI1640 media was added to the buffy coat andcentrifuged at 300×g for 10 minutes at room temperature. The cell pelletwas resuspended in 10 ml of red blood cell lysis solution (SigmaAldrich) and incubated for 5 minutes at room temperature. Cells werewashed once with media and centrifuged as previously described. Volumewas brought to 40 ml with Phenol red free-10% RPMI1640 media and cellswere counted using Vicell cell counter and viability analyzer (BeckmanCoulter).

Isolation of Primary Human CD4+CD25− T Effector Cells

Human CD4+CD25 cells were further purified from PBMC via two-stepmagnetic beads based isolation procedure using human CD4+CD25+Regulatory T Cell Isolation kit (Miltenyi Biotec). First, PBMC cellswere incubated with biotin-antibody cocktail at 4° C. for 5 minutes andsubsequently incubated anti-Biotin microbeads for 10 minutes. This stepwas to label the non-CD4+ T cells. Cells were then passed through a LDcolumn in the magnetic field of a MidiMACS separator. The effluent whichis the unlabeled pre-enriched CD4+ cell fraction was collected andincubated with CD25 MicroBeads at 4° C. for 15 minutes. The labelledcells were passed through a MS column in the magnetic field of aMiniMACS Separator. The flow-through containing the unlabeled CD4−CD25−T effector cells was collected for downstream activation assays.

Isolation of Primary Human CD4+ T Cells Directly from Blood

Human CD4+ T cells were isolated directly from fresh human blood usingHuman CD4+ T cells Enrichment Cocktail (Stem Cell Technologies).RosetteSep Human CD4+ T Cell Enrichment Cocktail (50 μL/mL) was added towhole blood and mixed well. After 20 minutes of incubation at roomtemperature, an equal volume of PBS+2% FBS was added with gentle mixing.The diluted sample was layered on top of the density medium andcentrifuged for 20 minutes at 1200×g at room temperature with the brakeoff. The enriched cells from the density medium:plasma interface werecarefully poured into a new conical tube. Next the red blood cells werelysed with Red Blood Cell Lying Buffer (Sigma Aldrich) and the enrichedcells were washed with PBS+2% FBS twice. The CD4+ T cells wereresuspended in 40 ml of with PBS+2% FBS and counted with Vi-Cell cellcounter.

Experimental Protocols Human CD4+CD25− T Effector Cell In VitroActivation Assay—Bound Assay.

Non tissue culture treated 96 well flat bottom plates were coated with100 μl/well of coating buffer (Biolegend) containing 1 μg/ml anti humanCD3 antibody (eBioscience) and various testing antibodies overnight. Onthe next day, the pre-coated plates were washed three times with 10% FBScontaining RPMI-1640 medium. Human CD4+CD25− T effectors cells wereisolated and labelled with CFSE as described and seeded onto the plates.After incubating at 37° C. for 2.5 days, cells were harvested andanalyzed by flow cytometry for proliferation and activation markerexpression. Cell culture supernatants were also collected for multiplexcytokines measurement by Meso Scale Discovery (MSD).

CFSE Proliferation Assay

Cells to be labelled were resuspended in 1 ml of pre-warmed PBS at afinal concentration of up to 1E7 cells/mL in a 15 ml of conical tube.One microliter of 2 mM stock CFSE solution (Life Technologies) was addeddirectly into the cells followed by immediately vortexing to ensureuniform labelling. After incubating at room temperature for 5 minutes,the staining was quenched by adding 14 ml of ice-cold cell culturemedia. The labeled cells were washed three times with ice-cold media.Cells were counted and adjusted to 1E6 cells/ml in RPMI1640+10% FBSsupplemented with 100 IU/ml of IL-2 (PeproTech) and seeded on anti CD3and testing antibodies coated plates. After T cell activation, cellswere harvested and washed with PBS+0.5% BSA once before proceeding tomulti-color staining step for flow cytometry analysis.

Multi-Color Flow Cytometry

Activated T cells were harvested and washed with PBS. Cells were firststained with LIVE/DEAD Fixable Far Red cell viability dye (LifeTechnologies) following vendor's protocol. After washing the dye off,detection antibodies conjugated with different colors were incubatedwith cells at 4° C. for 30 minutes. Stained cells were washed once withice cold FACS staining buffer (PBS+0.5% BSA) before running on FACSCanto or FACS Canto II flow cytometer. Cytometer performance was checkeddaily using Cytometer Setup & Tracking beads (BD Biosciences) and PMTvoltages and area scaling were set based on unstained cells.Compensations were performed using OneComp or UltraComp beads(eBioscience) that were individually stained with detection antibodiesconjugated with each fluorochrome.

MSD Cytokine Analysis

IFN-γ, IL-10, IL-2 and TNF-α cytokine levels in the tissue culturesupernatant were determined using MSD human V-Plex customized kit.Samples were first diluted 1:200 in Diluent2. Calibrators were alsoprepared in Diluent2 following kit manual. Diluted samples andcalibrators were added to black MSD plate which was subsequently sealedwith an adhesive plate seal and incubated at room temperature withshaking for 2 hours. After adding 25 μL of detection antibody solutionwhich was freshly prepared in Diluent2 to each well, the plate wasre-sealed and incubated at room temperature with shaking for another 2hours. The plates were washed 3 times with 150 μL/well of PBS plus 0.05%Tween-20 before adding 150 μl/well of freshly diluted 2× read buffer andimmediately read on MESO QuickPlex reader. Data were analyzed using MSDWorkbench software.

Human CD4+ T Cells In Vitro Activation Assay—Bound and Soluble

Freshly isolated human CD4+ T cells were pre-stimulated on 24 wellplates coated with anti-CD3 (1 μg/ml) and anti-CD28 (3 μg/ml) for 48hours. Cells were harvested, washed and mixed with anti-CD3 DynaBeads(Life Technologies) at 1:1 ratio in AIM-V medium supplemented with 100IU/ml of IL-2 (PeproTech). Cells/beads mixture were then seeded at 100 kper well onto 96 well flat bottom plates either non-coated (for solubleformat) or coated with H2L5 hIgG4PE (for bound format). For the solubleformat, H2L5 hIgG4PE was added to the wells at the time of cell seeding.After incubating at 37° C. for 3.5 days, cell culture supernatants werecollected for multiplex cytokines measurement by MSD.

Soluble Human PBMC In Vitro Activation Assay

Freshly isolated human PBMCs were pre-stimulated on 24 well platescoated with anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) for 48 hours.CFSE stained cells were prepared and mixed with anti-CD3 DynaBeads (LifeTechnologies) at 1:1 ratio in AIM-V medium supplemented with 100 IU/mlof IL-2 (PeproTech). Cells/beads mixture were then seeded at 200 k perwell onto 96 well plates that were pre-coated with 1 μg/ml of anti CD3antibody. H2L5 hIgG4PE and control antibody was added directly to thewells in their soluble form. After incubating at 37° C. for 3.5 days,cell culture supernatants were collected for multiplex cytokinesmeasurement by MSD, and cells were harvested for proliferation andmarker expression analysis by flow cytometry.

Data Analysis Flow Cytometry Data Analysis

Flow data was analyzed by FlowJo software (FlowJo LLC). Dead cells werefirst gated out based on LIVE/DEAD cell viability dye staining. Doubletswere gated out on FSC-H:FSC-W scatter plot. The resulted live singlecells were analyzed for activation marker expression within different Tcell sub-populations such as CD4+ or CD8+ T cells and reported aspercentage of parent population or Median Fluorescent Intensity (MFI).

CFSE Proliferation Analysis

CFSE data were also analyzed by Flowjo. After excluding the dead cellsand the doublets, a “proliferated cell” gate was drawn based on nonactivated T cells. Any cells fall in this gate in any given sample werecounted as proliferated cells. Data were reported as percentage ofproliferation.

Cell Depletion Analysis by FACS

Cell depletion was analyzed by FlowJo. First, a live cells gate wasdetermined based on LIVE/DEAD cell viability dye staining. Then thedoublets were gated out as previously described. The percentages of liveCD4+ or CD8+ T cell sub-population were calculated as an indicator forcell depletion.

Antibody Dose Response Curve Fitting Analysis

The dose response data were imported into GraphPad Prism software andtransformed into log scale. Agonist dose response with various slopemodel was used to curve fit the data and generate EC50 values. Thefitting formula is listed below:

Y=Bottom+(Top−Bottom)/(1+10̂((LogEC50−X)*HillSlope))

Results Lead Murine Anti Human ICOS Antibodies Identification

Fourteen murine mAbs were screened for human and cynomolgus ICOS bindingand agonist activity. Twelve were able to be re-cloned, sequenced, grownup and purified in sufficient amounts for functional studies. All weretested for binding characteristics using BIAcore. Two were found to bevery weak/non-binders. Ten purified mAbs tested by functional “agonism”analysis. The four best agonist mAbs (designated as 422.2, 279.1, 314.8and 88.2 in Table 5 below), based upon their ability to induce T cellproliferation and IFN-γ cytokine production across multiple healthyhuman donors, were selected and made as human IgG1 chimeras. The CDRsequences for 314.8, 88.2, 92.17, 145.1, and 53.3 are shown with otherICOS mAbs in PCT/EP2012/055735 (WO 2012/131004).

The heavy chain variable region for clone 88.2 is presented below as SEQID NO:13:

(SEQ ID NO: 13) QVQLQQPGAELVRPGASVKLSCKASGYSFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQMFKDKATLTVDKSSNTAYMQLTSPTSEDSAVYYCTRWNLSYYFDNNYYLDYWGQGTTLTVSSThe light chain variable region for clone 88.2 is presented below as SEQID NO:14:

(SEQ ID NO: 14) QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYNNHLVF GGGTKLTVL

TABLE 5 ICOS mAb Binding and Competition Com- Competi- peti- ICOSBiacore Biacore CD28/ ICOS-L tion tion mAb human cyno CTLA4 bindingagainst against Clone # (nM) (nM) x-reactive inhibition 314.8 121.4 53.330.4 19.7 − ++ + + 88.2 31 23 − + + + 92.17 27.5 18.8 − ++ + + 145.149.5 43.5 − ++ + + 314.8 17 9.3 − +++ + + 121.4 15 58 − − −/+ −/+ 202.2446 19 − + + + 279.1 39 33 − + + + 293.1 7.6 10 − ++ + + 422.2 5.7 4.46 −++ ++ +

Eight of the best ICOS binders were tested for IFN-γ production and Tcell proliferation using a cell based assay in a dose escalation designagainst mouse IgG1 and EPR7114 controls. Based on these assays, clonedesignated as 422.2 was selected for humanization. See FIGS. 1 and 2.

Example 3 Antibody Humanization Experimental Protocol(s)

Recovery of Antibody Variable Genes from mRNA and Generation of ChimericFc Wild-Type Antibody

Total RNA was purified from the 422.2 hybridoma cell pellet, reversetranscribed to generate cDNA from which the variable gene products,approximately 400 bp, were isolated by PCR and purified by agarose gelelectrophoresis.

The purified variable region fragments were cloned in to pTT5 vectorscontaining either the human IgG1 constant region or the human Kappaconstant region and transformed into DH5α competent cells. Colonies werepicked and used to inoculate L-broth containing ampicillin. Plasmid DNAwas isolated from the cultures using a QuickLyse mini-prep kit. Variableheavy and light chain genes were sequenced and sequence data was alignedby informatics to identify the variable heavy and light chain genesequences.

Cloning of Codon Optimised Chimeric 422.2 Antibodies

The mature murine variable region protein sequences were reversetranslated to DNA then codon optimised. The V_(H) and V_(L) sequenceswere then modified to include the preferred five prime untranslatedregion and preferred cloning sites at either end. The adapted V_(H)sequence was constructed de novo using a PCR-based strategy andoverlapping oligonucleotides then grafted onto human IgG1 Fc wild typeor Fc disabled hIgG1 (L235A, G237A) or hinge stabilised hIgG4 (S228P,L235E) (IgG4PE) present in pTT vectors. The adapted V_(L) sequence wasconstructed de novo using a PCR-based strategy and overlappingoligonucleotides then grafted onto a kappa constant region present in apTT5 vector.

-   The resulting pTT plasmids were used in HEK transfections to produce    the chimeric antibodies

Humanization of the Variable Domains of 422.2

Human variable (V) gene templates were chosen for humanization of 422.2by searching appropriate in-house human germline heavy and light chaindatabases with CDR-masked 422.2 V regions using BLASTP. IGHV1-69 andIGKV3-11 were chosen from the top BLASTP hits as the V gene frameworktemplates for 422.2 humanization. IGHJ6 and IGKJ2 human germline Joining(J) genes were chosen for humanization of 422.2.

Residue differences between the chosen human germline genes and the422.2 sequence were identified to aid in the selection of back-mutations(mutations made to change the specific human framework residue to themurine residue). Six humanized VH variants and six humanized VL variantswere designed, codon optimised and then modified to include preferred 5′and 3′ extensions. The adapted variable region sequences wereconstructed de novo using a PCR-based strategy and overlappingoligonucleotides then respectively cloned into pTT vectors.

The resulting pTT plasmids were used in HEK transfections to produce thehumanized antibodies. HEK2936E suspension cells were counted and dilutedto 1.5×10⁶ cells/mL to 2×10⁶ cells/mL using Freestyle expression medium293 supplemented with 0.05% Geneticin and for some experimentssupplemented with 1% pluronic F68. DNA and transfection reagent (Geminior 293-Fectin) were added to OptiMEM and gently homogenised prior toincubation for 20 to 30 minutes at room temperature. The DNA-lipidcomplexes were then combined with the cell suspensions and thetransfected cells were incubated at 37° C., 5% CO2, 125 rpm. For sometransfections, a tryptone feed (Freestyle expression medium 293supplemented with 1% pluronic F68 and 20% w/v casein tryptone) was addedto each transfection 24 to 48 hours after transfection. Transfected cellsuspensions were incubated for 5 to 8 days until viable cells droppedbelow 60% then centrifuged (construct dependent). Supernatants wereharvested and filtered.

Antibodies were purified by passing supernatants through a 1 mL HiTrapProtein A HP column to enable antibody capture, washing the columnthrough with 10 mL of PBS and eluting with 5 mL of IgG Elute (Pierce,21009). Purified protein was buffer exchanged into PBS using theMillipore Centricon® Centrifugal Filter Units (30K cut-off) andquantified on the Nandrop spectrophotometer.

Results Constructed Expression Plasmids

The murine antibody variable gene sequences of hybridoma clone 422.2were successfully recovered and the sequences are shown below as SEQ IDNOs: 19 and 20, respectively as well as in FIG. 8.

422 HC (SEQ ID NO: 19)QVQLQQSGPELVRPGESVKISCMGSGYTFTDYAMHWVKQSHAKSLEWIGLISIYSDHTNYNQKFQGKATMTVDKSSSTAYMELARLTSEDSAIYYCGRNN YGNYGWYFDVWGAGTTVTVSS422 LC (SEQ ID NO: 20)ENVLTQSPAIIVISASPGEKVTMTCSASSSVSYMHWYQQKSITSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFG GGTKLEIKR

Cloning the recovered variable regions into selected pTT5 vectorsresulted in generation of plasmids encoding the chimeric light chainsequence of 422.2 and the chimeric heavy chain sequences on hIgG1 Fcwild type, hIgG1 Fc disabled (L235A/G237A, EU numbering) or a hIgG4 PE(S228P/L235E, EU numbering). The chimeric antibodies were used toconfirm functionality of the cloned mouse V-regions and to identify themost suitable isotype.

Construction of pTT mammalian expression plasmids encoding the heavy andlight chains of the various humanized variants of 422.2 was carried outsuccessfully. Expression, purification and identification of H2L5hIgG4PE

The mature protein sequences of H2L5 hIgG4PE have been included withadditional labeling in FIG. 9. The DNA sequences of the coding regionsof H2L5 hIgG4PE heavy and light chains have been included in FIGS. 10and 11.

Example 4 Functional Analysis of H2L5 Fc Receptor Binding

Humanized antibody H2L5 was modified from a human IgG1 isotype to amodified human IgG4 isotype incorporating mutations S228P, L235E (EUnumbering) to prevent antigen binding fragment (Fab) arm exchange. HumanIgG4PE was selected over human IgG1 as it diminishes binding of the mAbto activating Fcγ receptors and C1q, therefore, reducing the potentialof the mAb to induce depletion of ICOS positive cells byantibody-dependent cytotoxicity (ADCC) or complement dependentcytotoxicity (CDC). In addition, human IgG4PE (S228P, L235E) retainsbinding to FcγRIIb (inhibitory Fcγ receptor). Interaction with FcγRIIbmay be critical for the agonistic activity of ICOS antibodies byenabling antibody cross-linking. Interaction with FcγRIIb has been shownto be critical for the agonistic activity of other immunomodulatoryantibodies targeting TNF-α family receptors as well as CD28(Bartholomaeus P et al., “Cell contact-dependent priming and Fcinteraction with CD32+ immune cells contribute to the TGN1412-triggeredcytokine response.” J. Immunol., 192(5); 2091-8 (2014)).

It was further shown that human IgG4PE diminishes binding of the mAb toactivating Fcγ receptors (FcγRI, FcγRIIa and FcγRIIIa) and C1q,therefore reducing the potential of the mAb to induce depletion of ICOSpositive cells by antibody-dependent cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC). In addition, human IgG4PE (S228P, L235E)retains binding to FcγRIIb (inhibitory Fcγ receptor) (Table 6).

Table 6 below displays representative binding affinities to human Fcγreceptors of the lead H2L5 as either a hIgG1 or a hIgG4PE antibody.

TABLE 6 Representative Affinities of Lead Humanized ICOS antibody, aseither a hIgG1 or a hIgG4PE, to human Fcγ receptors Fc 

 R Fc 

 R IIIa Fc 

 R IIa Fc 

 R IIa IIIa F158 Fc 

 R I H131 R131 Fc 

 R IIb V158 KD Antibodies KD (nM) KD (nM) KD (nM) KD (nM) KD (nM) (nM)422 H2L5 IgG1 60.8 405 662 1340 281 862 WT 422 H2L5 645 NB 2500 1470 NBNB hIgG4PE (H2L5 IgG4PE) NB = no binding.

Experimental Protocol Functional Evaluation of 422.2 Humanized Variants

To humanize the four candidate ICOS agonist antibodies, mouse-humanchimeras, which are fusions of mouse V region and human IgG1 Fc portion,were generated. These four chimera antibodies were tested in human wholePBMC activation assay as plate bound form. Anti-ICOS chimera 422.2showed the best agonistic activity in the bound PBMC activation assay.Combined with binding data and biophysical properties, 422.2 clone waschosen for humanization. Four humanized 422.2 variants were selectedbased on ICOS binding and biophysical characteristics (422.2 H2L0, H2L5,H5L0 and H5L5) and were tested in bound human PBMC activation assays.The H2L5 variant demonstrated comparable or better T cell activation asmeasured by cytokine production relative to other variants (FIG. 3).

The heavy chain (V_(H)) variable region and mature heavy chain for theH5 variant are presented below as SEQ ID NOs:15 and 16, respectively.

H5 VH (SEQ ID NO: 5) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWIGLISIYSDHTNYNQKFQGRATMTVDKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSS  Mature H5 heavy chain (SEQ ID NO: 16)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVRQAPGQGLEWIGLISIYSDHTNYNQKFQGRKTMTVDKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPLAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

The light chain (V_(L)) variable region and mature light chain for theL0 variant are presented below as SEQ ID NOs:17 and 18, respectively.

L0 VL (SEQ ID NO: 17) EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPYTFGQG TKLEIKMature L0 light chain (SEQ ID NO: 18)EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTIKSFNRGEC 

Selection of IgG4[PE] as the Isotype

The 422 H2L5 IgG1 was subsequently tested in various whole PBMCactivation assays in soluble form. This soluble format is likely to bemore relevant to the in vivo condition as the whole PBMC assays containlymphocytes, monocytes and other immune cells in the same well. However,422 H2L5 IgG1 mAb consistently showed decreased viability of T cellpopulations which is reminiscent of T cell depletion. This result wasobserved to different degrees in 11 healthy donors which were tested andwas more prominent in CD4+ T cells than in CD8+ T cells. In contrast,the 422 H2L5 antibody did not show a significant decrease in T cellviability when expressed as either an IgG4[PE] or Fc-disabled isotype,suggesting that the decreased viability may have been due toFcγR-mediated antibody-dependent cellular cytotoxicity (ADCC) (FIG. 4).

Dose Response of H2L5 hIgG4PE in CD4+ T Cell Activation Assay

To quantify the T cell activation effects of H2L5 hIgG4PE, human primaryCD4+ T cells were first pre-stimulated by plate bound anti-CD3 (1μg/ml)/anti-CD28 (3 μg/ml) for 48 hours to induce levels of ICOSreceptor on the surface of T effector cell populations, followed byre-stimulation with anti CD3 DynaBeads and H2L5 hIgG4PE. A 10-point doseresponse curve was generated by treating the pre-simulated CD4+ T cellswith serial concentrations of bound or soluble H2L5 hIgG4PE in thepresence of anti-CD3 DynaBeads. Results showed that both bound andsoluble H2L5 hIgG4PE increased IFN-γ and TNF-α cytokines productions ina dose dependent manner in two separate healthy human donors (FIG. 5). Adose-response curve fitting analysis was performed to generate EC50values. Interestingly, H2L5 hIgG4PE treatment resulted in asignificantly greater magnitude of cytokine induction when the antibodywas plate-fixed as opposed to being added as a soluble protein to thesupernatant of the T cell cultures.

Functional Testing of H2L5 hIgG4PE in Soluble Human PBMC ActivationAssay

In order to test the function of H2L5 hIgG4PE in whole human PBMC exvivo culture, PBMC from healthy human donors were prepared with platebound anti-CD3 and anti-CD28 for 48 hours, followed by soluble H2L5hIgG4PE treatment in the presence of anti CD3 dynabeads (bead:cell=1:1ratio) for 3.5 days. Cytokine production and T cell Granzyme Bexpression was examined as functional readouts for T cell function. Theresults from 3 donors were summarized in FIG. 6, which provide evidenceto suggest that H2L5 hIgG4PE induces proliferation, cytokine productionand increased cytotoxic potential in activated PBMCs from healthy humandonors (FIG. 6).

ICOS mAb Activity Against Pre-Stimulated Human PBMCs

The activity of H2L5 hIgG4PE was evaluated in a PBMC pre-stimulationassay in which the PBMC were pre-stimulated by plate bound anti-CD3(clone OKT3, 1 μg/ml) and anti CD28 (clone CD28.2, 3 μg/ml) for 48hours. Next, in order to identify the optimal pre-stimulationconditions, human CD3 Dynabeads and CD3/CD28 Dynabeads (ThermoFisher) atdifferent bead to cell ratios were used to pre-stimulate PBMCs. After 48hr pre-stimulation, cells were harvested and beads magnetically removedbefore re-stimulation with anti CD3 Dynabeads (bead to cell ratio=1:1)in the presence or absence of soluble ICOS mAb. H2L5 hIgG4PE ICOSagonist mAb resulted in induction of IFNγ as compared to isotype controlin all pre-stimulation conditions tested; however, the magnitude of IFNγproduction inversely correlated with strength of the pre-stimulation.

Example 5 T Cell Activation Markers Methods

Concentration-dependent changes in functional endpoints were assessed bytreating healthy human PBMCs with immobilized H2L5 hIgG4PE atconcentrations ranging from 0.1 μg/mL to 100 μg/mL concurrently withanti-CD3 antibody treatment at 0.6 μg/mL. Changes in expression of Tcell surface activation markers CD69, OX40 and CD25 were evaluated byflow cytometry and considered a measure of T cell activation.Proliferation of T cells was measured by the changes in Ki67 nuclearstaining. Change in levels of various Th1, Th2 and Th17 cytokines wereevaluated on the MSD platform in response to H2L5 hIgG4PE treatment inthe presence of CD3 engagement. The 24- and 48-hours post treatmenttimepoints were selected to ensure the capture of both early cytokinechanges as well as proliferation changes which are predominantly noticedat later time points.

Experimental Preparation(s) Isolation of Human Peripheral BloodMononuclear Cells (PBMCs)

Whole blood was collected from healthy donors with syringes coated withliquid sodium heparin (Sagent 10 IU/mL final concentration) andsubsequently diluted 1:1 with phosphate buffered saline (PBS). Dilutedblood (35 mL) was layered on top of 15 mL Ficoll density gradient medium(GE Healthcare) and centrifuged at 1200×g for 20 minutes at roomtemperature (RT) without brakes. The white mononucleated cell layer wascarefully transferred into a new 50 ml tube. An equal volume of PBS wasadded to the tube and centrifuged at 400×g for 5 minutes at RT. PBMCwere washed once with PBS and centrifuged as previously described. PBMCwere resuspended in 50 mL AIM-V media Cells were counted using a Vi-Cellcell counter and viability analyzer (BeckmanCoulter).

Antibody Coating

Anti-human CD3 antibody was diluted in coating buffer to a finalconcentration of 0.6 μg/mL. 100 μL diluted antibody was coated on96-well, flat-bottom plate overnight at 4° C. Next day, stock solutionsof 10.1 mg H2L5 hIgG4PE and 7.9 mg anti-RSV IgG4 PE isotype controlantibody were 1:2 serially diluted in coating buffer to give finalantibody concentrations ranging from 100 to 0.1 μg/mL. 100 μL dilutedantibodies were coated on anti-CD3 coated plate for 4 hours at roomtemperature.

Experimental Protocol(s) Human PBMC Activation Assay

H2L5 hIgG4PE was tested in a human PBMC activation assay where TCRengagement via anti-CD3 antibody and ICOS co-stimulation with H2L5hIgG4PE occurred concurrently, and the activation effects were monitoredat 24 and 48 hours post activation. This experiment was repeated fourtimes (n=4) with blood from four different donors. Two hundred (200) μLPBMCs (1×10⁶ cells/mL) in AIM-V medium were added into anti-CD3antibody-coated wells with various concentrations of H2L5 hIgG4PE orIgG4 isotype control. Three technical replicates were included for eachassay condition. PBMCs were cultured at 37° C. and 5% CO₂ for varioustimes as indicated above. Supernatants were collected at 24- and 48-hourtime points and then stored at −80° C. for analysis of secretedcytokines on the MSD platform. Cells were transferred into 96-deep wellplate at both 24 and 48 hours and washed twice with 1 mL FACS StainingBuffer stained with fluorophore-conjugated antibodies or isotypecontrols.

Cell Surface Staining

Cells were first stained with 100 μL of fixable viability dye eFluor 506pre-diluted 1:1000 in PBS for 30 minutes in the dark at 4° C. Cells werewashed and then incubated with detection antibodies to cell surfacemarkers conjugated to different fluorophores on ice for 30 minutes.Post-staining with cell surface antibodies, samples not to be stainedfor internalization markers were washed once with ice cold FACS StainingBuffer before running on FACS Canto II flow cytometer.

Cytometer performance was appropriately evaluated daily using theCytometer Set-up and Tracking beads. Compensations were performed usingAbC anti-mouse Bead kit that were individually stained with detectionantibodies conjugated with each fluorochrome. Samples were run and dataacquired after proper compensation set up was ascertained with a mix ofbeads mentioned above.

Intracellular Staining for Foxp3 and Ki67

Following the cell surface staining, the cells were fixed andpermeabilized for staining for intracellular markers. Using theTranscription Factor Buffer set, the Fixation/Permeabilization Bufferwas prepared by diluting this 1:3 in Diluent Buffer. The Perm WashBuffer was prepared by diluting the 5× Perm/Wash Buffer stock indeionized water. One (1) mL of Fix/Perm Buffer was added to each sample,and the plates were immediately vortexed on the shaker. The plates wereincubated in the dark at 4° C. for 45 minutes. Following centrifugation,1 mL of Perm/Wash Buffer was added, and the plates were mixed andcentrifuged for a total of two washes. The internalization cocktail wasprepared with marker antibodies. 100 μL of the internalizationantibodies were added to the appropriate samples, and the plates wereincubated in the dark at 4° C. for 30 minutes. Samples were washed twicewith 1 mL of Perm Wash Buffer, resuspended in 250 μL of flow buffer andrun on FACS Canto II flow cytometer.

Human Special Order 9-Plex Cytokine Assay

Cytokine levels were measured using the MSD Human Special Order 9-Plexkit.

Samples and calibrators were diluted in Diluent 43. Samples were diluted1:10 for the 9-plex assay and 1:200 for the IFNγ assay described in0.250 μL of the diluent was added to each of two calibrator panels.After vortexing, the calibrators were incubated on ice for at least 5minutes. Two hundred (200) μL of each calibrator panel was added to 400μL of diluent to make the top concentration of calibrator, and a 1:4serial dilution was used to prepare the 6 additional calibratordilutions. Diluent 43 was used as the plate background. 50 μL of dilutedsamples (in triplicate) and calibrators (in duplicate) were added to theMSD plate. Plates were sealed and incubated at RT with shaking for 2hours. Plates were washed 3 times with 150 μL of diluted MSD Wash Bufferfrom the kit. For each plate, detection antibody solution was preparedby combining 60 μL of each of the 9 detection antibodies brought up to 3mL with Diluent 3. Following the addition of 25 μL of detection antibodysolution, the plates were sealed and incubated at room temperature, inthe dark, with shaking for 2 hours. Plates were washed as above. 150 μLof 2× Read Buffer was added to the plates, and they were read on theQuickPlex.

Human IFNγ Cytokine Assay

Samples and calibrators were diluted in Diluent 2. 1 mL of the diluentwas added to the calibrator. After vortexing, the calibrator wasincubated on ice for 5 minutes. This is Calibrator 1. A 1:4 serialdilution was used to prepare the 6 additional calibrator dilutions.Diluent 2 was used as the plate background. Fifty (50) μL of dilutedsamples (in triplicate) and calibrators (in duplicate) were added to theMSD plate. Plates were sealed and incubated at RT with shaking for 2hours. Plates were washed 3 times with 150 μL of diluted MSD Wash Bufferfrom the kit. Detection antibody solution prepared in Diluent 3. Foreach plate, 60 μL of each of the detection antibody was added to thediluent for a total of 3 mL of detection reagent. Following the additionof 25 μL of detection antibodies, the plates were sealed and incubatedat room temperature, in the dark, with shaking for 2 hours. Plates werewashed 3 times. Read Buffer was added to the plates and they were readon the QuickPlex.

Data Analysis Cytokine and Flow Cytometry Data Analysis

Results of MSD cytokine assay were analyzed using MSD DiscoveryWorkbench software, version 4.0 (Meso-Scale), Microsoft Excel, andGraphpad Prism. Flow cytometry data were analyzed by DIVA, and numberswere plotted in GraphPad Prism software.

Antibody Dose-Response Curve Fitting Analysis

The dose response data were imported into GraphPad Prism software andtransformed into log scale. Agonist dose response with various slopemodel was used to analyze the data and generate EC50 values. The fittingformula is listed below:

Y=Bottom+(Top−Bottom)/(1+10̂((LogEC50−X)*HillSlope))

Statistical Analyses

Differences between H2L5 hIgG4PE and isotype antibody control values inthe proliferation study were analyzed for a statistical significance bytwo-paired Student's t test.

Results

Evaluation of Cytokine Changes with H2L5 hIgG4PE

Treating PBMCs with immobilized H2L5 hIgG4PE in the presence of anti-CD3induced the secretion, to different degrees, of Th1 cytokines such asIFNγ, TNFα, the Th2 cytokines, IL-6 and IL-10, as well as the Th17cytokine IL-17a in a concentration-dependent manner. Other cytokinesmeasured such as IL-4, IL-5 and IL-13 also showed a lesser extent ofconcentration-dependent response to H2L5 hIgG4PE stimulation. Theresults from four separate donors are summarized in Table 7.

Functional Evaluation of H2L5 hIgG4PE Activity on Cell Surface Markersof T Cell Activation by Flow Cytometry

H2L5 hIgG4PE treatment with concurrent CD3 stimulation in unactivatedhuman PBMC (n=4 donors) induced significant changes in T cell activationmarkers (Table 7 and 8)). Robust increases in CD25 and OX40 positive CD4and CD8 T cells were observed in H2L5 hIgG4PE treated samples whencompared to the human IgG4 isotope control samples. The percent of CD69positive CD4 and CD8 T cells were also increased at 24- and 48-hour timepoint in a concentration-dependent manner.

Characterization of the Effect of H2L5 hIgG4PE on T Cell Proliferation

Immobilized H2L5 hIgG4PE treatment with concurrent CD3 activationresulted in a concentration-dependent increase in both CD4 and CD8 Tcell proliferation (n=6 donors) as measured by intracellular Ki67staining (Table 8). H2L5 hIgG4PE also increased CD4+CD25+Foxp3+regulatory T cell proliferation in a concentration dependent manner butto a lesser extent than what was observed with total CD4 and CD8 Tcells. The enhancement of T cell proliferation by H2L5 hIgG4PE was onlyobserved at the 48 hour time point. The increased proliferation of theregulatory T cells was not significant whereas theconcentration-dependent increase in proliferating CD4+ cells (p<0.05 forconcentrations greater than 0.4 μg/mL H2L5 hIgG4PE) and CD8+ T cells(p<0.05 for concentrations between 0.2 and 1.6 μg/mL) was significant(see Table 7).

TABLE 7 EC50 values (μg/mL) from all functional endpoints for H2L5hIgG4PE in human PBMC activation assay. Donor_1136F50 Donor_185M45Donor_1124F36 Donor_1149M52 24 hr 48 hr 24 hr 48 hr 24 hr 48 hr 24 hr 48hr IL-10 1.7 1.3 0.7 0.8 0.7 0.6 0.5 0.6 IFN-γ 0.4 1.5 0.3 0.7 0.3 0.50.2 0.6 IL-17a 1.4 1.6 0.8 1.1 0.6 0.8 0.7 1.0 IL-6 0.7 1.1 0.7 0.8 NANA 0.2 0.3 TNF-α NA 0.3 NA 0.5 NA 0.3 NA 0.2 CD4+CD69+ 0.5 0.4 0.8 NA1.1 1.1 0.5 0.4 CD4+CD25+ 0.3 0.6 2.4 0.5 0.6 0.6 0.6 0.4 CD4+OX40+ 0.20.4 1.6 NA 0.6 1.2 0.6 0.4 CD8+CD69+ 0.5 0.6 1.2 0.5 0.8 1.1 0.6 0.4CD8+CD25+ 0.4 0.7 2   0.5 0.6 0.5 1.0 0.4 CD8+OX40+ 0.3 0.5 1.6 NA 2.40.5 1.5 0.3 CD4+Ki67+ NT NT NA 0.6 NA 0.8 NA 0.5 CD8+Ki67+ NT NT NA 0.4NA 0.7 NA 0.3 NA = no analysis (EC50 values not accurate due to poorcurve fitting). NT = no tested.

TABLE 8 Percent of CD25, Foxp3 and Ki67 positive CD4 or CD8 T cells inhuman PBMCs after stimulation with H2L5 hIgG4PE in the presence ofanti-CD3 for 48 hours H2LA hIgG4PE Isotype Control Antibody (% of CD4 orCD8 T cells) (% of CD4 or CD8 T cells) TTEST concentration Donor DonorDonor Donor Donor Donor (p μg/ml 185M45 1124F36 1149M52 185M45 1124F361149M52 value) % Treg 0 10.2 3.8 6.1 10.2 3.8 6.1 0.415 CD4 0.1 12.4 4.27.6 8.6 3.6 6.6 0.220 T cells 0.2 11.8 4.5 9.8 8.5 3.2 7.1 0.054 0.414.3 5.2 11.3 8.6 3.4 7.4 0.076 0.8 18.6 6.9 14.3 8.4 3.4 7.3 0.069 1.621.4 7.5 15.9 7.7 3.8 7.2 0.095 3.1 20.8 7.9 15.0 10.4 3.9 7.1 0.058 6.318.7 8.3 14.7 10.3 3.6 8.3 0.026 12.5 20.2 7.6 15.1 9.6 4.0 8.1 0.072 2519.5 7.6 13.9 11.5 5.1 8.3 0.079 50 20.0 7.9 13.3 9.6 5.0 9.7 0.143 10018.7 7.9 13.3 11.5 5.6 8.8 0.080 % 0 7.2 1.2 2.3 7.2 1.2 2.3 0.693 Ki67+0.1 9.6 1.5 2.9 6.1 0.9 2.1 0.219 CD4 T 0.2 9.0 2.1 4.4 5.0 0.9 2.40.098 cells 0.4 11.9 3.9 7.5 6.8 1.0 2.7 0.025 0.8 18.2 7.1 12.4 7.6 1.32.5 0.028 1.6 19.5 8.9 15.9 5.4 0.8 3.2 0.024 3.1 21.1 10.7 14.4 9.1 1.22.9 0.005 6.3 18.7 12.6 17.1 8.1 1.1 3.2 0.007 12.5 22.2 12.2 16.5 7.41.5 3.4 0.008 25 20.7 11.6 16.1 9.3 1.7 5.2 0.002 50 21.2 12.8 14.3 7.52.4 7.3 0.034 100 21.8 12.0 14.0 9.4 2.8 4.7 0.010 % 0 11.0 4.4 2.7 11.04.4 2.7 0.841 Ki67+ 0.1 15.2 8.3 4.8 9.6 5.3 2.3 0.061 CD8 T 0.2 13.510.9 7.8 8.2 3.8 2.9 0.014 cells 0.4 17.4 14.4 11.3 10.1 3.4 4.3 0.0230.8 20.7 19.6 13.8 14.2 4.5 2.0 0.047 1.6 22.2 22.8 16.1 10.7 4.0 3.70.025 3.1 21.8 26.7 13.6 14.5 4.4 3.0 0.099 6.3 20.2 29.8 16.0 10.8 3.35.0 0.103 12.5 21.7 29.6 15.6 10.7 4.1 3.4 0.074 25 20.0 28.7 14.6 10.46.1 5.0 0.084 50 19.0 31.4 13.5 7.6 5.1 5.5 0.114 100 19.2 27.4 13.4 7.97.1 3.5 0.051 % Ki67+ 0 29.7 10.2 11.3 29.7 10.2 11.3 0.920 Treg 0.132.8 12.5 14.4 32.4 10.0 12.3 0.127 cells 0.2 33.0 14.8 19.0 30.3 8.313.4 0.051 0.4 33.6 22.2 22.2 28.4 10.8 14.1 0.044 0.8 37.0 26.3 26.336.7 8.5 14.4 0.191 1.6 35.0 27.7 28.4 26.8 7.7 16.3 0.061 3.1 38.3 30.227.2 33.9 9.6 17.4 0.135 6.3 33.9 32.5 26.1 36.7 8.7 16.5 0.315 12.537.3 36.8 26.2 31.2 15.1 17.2 0.125 25 36.3 33.0 28.6 33.3 13.7 28.10.326 50 37.7 33.6 24.2 32.9 21.1 29.5 0.518 100 40.1 35.8 28.6 36.321.5 26.4 0.216

Discussion

It is well established that ICOS is important for T cell activation andinduction of both Th1 and Th2 cytokines. In this study, the in vitroactivity of H2L5 hIgG4PE (anti-ICOS agonist antibody), was demonstratedwith various measures of T cell activation and cytokine induction. Allmeasured T cell activation markers, CD25 (IL-2 receptor alpha-chain),CD69 (early activation marker) and OX-40 (co-stimulatory marker) wereupregulated upon treatment with H2L5 hIgG4PE in conjunction with CD3stimulation. Among the activation markers monitored, the percent of Tcells expressing CD69 and OX40 were strongly increased by H2L5 hIgG4PEtreatment. CD69 is an early activation marker and hence the effects arepredominant in the 24-hour samples. CD25, another important T cellactivation marker, was increased upon treatment with H2L5 hIgG4PE atboth time points, suggesting that H2L5 hIgG4PE plays an important rolein maintaining activation of T cells. Ki67 is a nuclear proteinassociated with cell proliferation. Flow cytometry data with Ki67intracellular staining indicated that immobilized H2L5 hIgG4PEsignificantly enhanced both CD4 and CD8 T cell proliferation in thecontext of TCR engagement. Though proliferation of regulatory T cellswas also increased by H2L5 hIgG4PE, the changes were not statisticallysignificant.

Human Th17 cells are a key player in the regulation of anti-tumorimmunity [Nunez, S., et al., T helper type 17 cells contribute toanti-tumour immunity and promote the recruitment of T helper type 1cells to the tumour. Immunology 2013; 139: 61-71]. Studies show ICOS isinvolved in human Th17 development and function [Kimura, A., et al.,Regulator of Treg/Th17 balance. Eur. J. Immunol. 2010; 40: 1830-1835;Paulos C M et al. The inducible costimulator (ICOS) is critical for thedevelopment of human Th17 cells. Sci Transl Med. (2010) 2(55); 55ra78;Nelson, M. H., et al. The inducible costimulator augments Tc17 cellresponses to self and tumor tissue. J Immunol, 2015; 194: 1737-1747]. Inthe current functional evaluation of H2L5 hIgG4PE, a majority ofcytokines related to inflammatory and immune responses were measured inthe supernatant of cell cultures after anti-CD3 and H2L5 hIgG4PEstimulation. H2L5 hIgG4PE strongly induces secretion of Th1 cytokines,IFN-γ and TNF-α and Th17 cytokine, IL-17a, in human PBMCs, suggestingthat H2L5 hIgG4PE has the potential to play an important role inanti-tumor responses. IL-6, together with TGF-β, is an importantcytokine for induction of the Th17 cell development from naive T cells.In contrast, IL-6 inhibits Treg differentiation induced by TGF-β[Kimura, A., 2010; Korn, T., et al., IL-6 controls Th17 immunity in vivoby inhibiting the conversion of conventional T cells into Foxp3⁺regulatory T cells. PNAS, 2008; 105: 18460-18465]. In this study, H2L5hIgG4PE was found to increase the secretion of IL-6 which may furtherenhance Th17 cell development. Agonist antibodies of T cell receptorssuch as CD28 and TNF receptor family members have been shown to producea bell shaped dose response curve [White, A. L., et al., Conformation ofthe Human Immunoglobulin G2 Hinge Imparts Superagonistic Properties toImmunostimulatory Anticancer Antibodies. Cancer Cell, 2015, 27: 138-148;Luhder, F., et al, Topological Requirements and Signaling Properties ofT Cell-activating, Anti-CD28 Antibody Superagonists. J. Exp. Med. 2003:955-966; Stebbings, R., et al., “Cytokine Storm” in the Phase I Trial ofMonoclonal Antibody TGN1412: Better Understanding the Causes to ImprovePreClinical Testing of Immunotherapeutics J. Immunol., 2007, 179:3325-3331; Rogers P R and Croft M, CD28, Ox-40, LFA-1, and CD4Modulation of Th1/Th2 Differentiation Is Directly Dependent on the Doseof Antigen. J Immunol 2000 164:2955-2963;doi:10.4049/jimmunol.164.6.2955]. H2L5 hIgG4PE also demonstrates asimilar hyperbolic functional response curve. This information is animportant component in ascertaining the best dose range of the antibodyto be used for optimal pharmacodynamic response.

Overall, H2L5 hIgG4PE, in conjunction with CD3 stimulation, was shown toenhance T cell activation, proliferation and proinflammatory cytokineinduction in line with its role as a potent activator of a T cellco-stimulatory receptor.

Example 6 Binding of Anti-ICOS Antibodies

The humanization protocol (Example 3) produced 36 heavy and light chainvariants which were screened for binding to human and cynomolgus ICOSwhilst also ensuring that they did not bind to human CD28 or CTLA-4. TheH2L5 variant was identified as the having high affinity for human andcynomolgus ICOS (1.34 and 0.95 nM respectively) whilst containing theminimum number of back mutations.

Changing the isotype of 422 H2L5 from IgG1 to IgG4PE does not affect theantigen binding of the antibody as H2L5 hIgG4PE has an affinity of 1.3nM to human ICOS. Concentration based inhibition of ICOS/ICOS-L bindingby H2L5 hIgG4PE is shown in FIG. 7.

Experimental Protocols

Binding of H2L5 hIgG4PE to Human ICOS

The binding kinetics affinity of the humanized H2L5 hIgG4PE antibody wasdetermined used the BIAcore T200Anti-human IgG on Fc2 of a CM5 chipAnti-ICOS H2L5 hIgG4PE captured on the surface. Anti-human IgG surfaceblocked with 0.1 mg/mL hIgG1 control to prevent non-specific binding ofrabbit Fc. Human and cyno ICOS (rabbit Fc) passed over the capturedantibodies at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. Buffer alone was usedto double reference binding curves. MgCl₂ was used to regeneratesurface. Run carried out at 25° C. Data fitted to 1:1 model using T200evaluation software. Antibody concentration: 2.5 μg/mL

Results

Binding of H2L5 hIgG4PE to Human and Cynomolgus ICOS

The binding kinetics affinity of the humanized H2L5 hIgG4PE antibody wasdetermined using the BIAcore T200.

The ICOS binding data was fitted to a 1:1 kinetics model using the T200data analysis software.

The binding affinity of H2L5 hIgG4PE for human ICOS is 1.34 nM andcynomolgus ICOS is 0.95 nM (see Table 9). These values are comparableand show, as expected, that a change to the Fc region of the moleculehas not affected the binding to the ICOS antigen. Table 9 shows Ka/Kd/KDfor humanized 422 (H2L5) IgG4PE to human and cynomolgus ICOS.

TABLE 9 Binding to human ICOS Sample Target Ka (1/Ms) Kd (1/s) KD (M)422 H2L5 Human ICOS-Fc 2.97E+05 3.96E−04 1.34E−09 IgG4PE 422 H2L5 CynoICOS-Fc 3.91E+05 3.71E−04 9.49E−10 IgG4PE

Discussion

As shown in Example 1 murine clone 422-2 was identified as the leadanti-human ICOS murine antibody. Humanization of this antibody produced36 heavy and light chain variants which were screened for binding tohuman and cynomolgus ICOS whilst also ensuring that they did not bind tohuman CD28 or CTLA-4. The H2L5 variant was identified as the having highaffinity for human and cynomolgus ICOS (1.34 and 0.95 nM respectively)whilst containing the minimum number of back mutations.

Changing the isotype from IgG1 to IgG4PE does not affect the binding ofthe antibody to ICOS.

Example 7 Binding of H2L5 hIgG4PE to Human Activated T Cells MethodsExperimental Preparation(s) CD3 Negative Isolation:

CD3+ T Cells were Negatively Isolated by Stemcell Rosette Sep Human TCell Enrichment Kit

Rosette Sep Human T Cell Enrichment:

100 mL fresh, whole blood was collected with syringes coated in liquidsodium heparin (Sagent 10 IU/mL final concentration). The blood fromeach collection tube was combined into a flask where 50 μL of RosetteSep Human T Cell Enrichment cocktail was added per ml of blood. (5mL/100 mL Donor Blood). The whole blood/Rosette Sep antibody cocktailwas incubated for 20 minutes at room temperature (RT). The Blood/RosetteSep antibody cocktail was then diluted 1:1 with 1× phosphate bufferedsaline (PBS)+2% FBS (fetal bovine serum). FBS for a final volume of 200mL. Next, 25 mL of diluted blood/antibody cocktail was then layered over15 mL of Ficoll gradient in Sepmate tubes (8 tubes in total for eachDonor). Loaded Sepmate tubes were then centrifuged at 1200×g for 20minutes RT with the brakes on. The top layer of plasma down to theperipheral blood mononuclear cells (PBMC) interface was taken off with apipette and discarded. The remaining plasma and buffy coat interfacewere decanted from Sepmate tubes into 50 mL conical centrifuge tubes (4tubes total). The tubes were topped off to 50 mL with PBS+2% FBS. Thecells were centrifuged at 400×g for 10 minutes at RT. Supernatants werediscarded. The pellets from each donor were then combined into a single50 mL conical tube, re-suspending the pellets in a total volume of 50 mLPBS+2% FBS. Cells were centrifuged at 400×g for 5 minutes at RT.Supernatants were discarded and the cell pellets were re-suspended in 2mLs of RPMI Complete Media (RPMI 1640+10% FCS+1 mM sodium pyruvate+2 mML-glutamine+penicillin 100 U/mL+streptomycin 100 μg/mL). Recovered CD3Cells were counted on the ViCell instrument and further diluted to1.2×10⁶ cells/mL. 1×10⁶ recovered cells were stained for CD3 PE-Cy7 toconfirm the quality of the T cell isolation.CD3+ T Cells were Negatively Isolated by Invitrogen Untouched T CellIsolation Kit PBMC Isolation:Briefly, 100 mL fresh, whole blood was collected from each donor withsyringes coated in liquid sodium heparin (Sagent 10 IU/mL finalconcentration). Blood was diluted (1:1) to final volume of 200 mL withPBS with 2% FBS. Twenty five (25) mL of diluted blood was layered over15 mL of Ficoll gradient in Sepmate tubes (8 tubes in total for eachdonor). Loaded Sepmate tubes were then centrifuged at 1200×g for 20minutes at RT with the brakes on. The top layer of plasma down to thePBMC interface was taken off with a pipette and discarded. The remainingplasma and buffy coat interface were decanted from Sepmate tubes into 50mL conical centrifuge tubes (4 tubes total). The tubes were topped offto 50 mL with PBS+2% FBS. The cells were centrifuged at 400×g for 10minutes at RT. Supernatants were discarded. The pellets from each donorwere then combined into a single 50 ml conical tube and re-suspended ina total volume of 50 mL PBS+2% FBS. Cells were centrifuged at 400×g for5 minutes at RT. Supernatants were discarded, and the cell pellets werere-suspended in an arbitrary volume of Isolation Buffer (dependent uponthe size of the cell pellet) provided in the Invitrogen Untouched T cellkit. Isolated PBMC's were then counted on the ViCell instrument andbrought to a final concentration of 1×10⁸ cells/mL in Isolation Buffer.

Invitrogen Dynabead Untouched Human T Cell Isolation:

2×10⁸ isolated PBMCs (2 mLs), 400 μl of FBS and then 400 μl of AntibodyMix from the Invitrogen Untouched T cell kit were added to each 15 mLtube and incubated for 20 minutes at 4° C. Cells were washed with 10 mLsof Isolation Buffer and centrifuged at 350×g for 8 minutes at 4° C.Supernatants were discarded, and the pellets were re-suspended in 2 mLsof Isolation Buffer. Next, 2 mLs of pre-washed Depletion Dynabeads wereadded to each tube. Cells were incubated with the beads for 15 minutesat room temperature with gentle tilting and rotation. Following the beadincubation, 10 mLs of Isolation Buffer was added and the cell/beadsuspensions were pipetted up and down 10 times. The tubes were placedinto a magnet for 2 minutes at room temperature. Without disturbing themagnetized beads, the supernatants containing the untouched T Cells werecollected. The beads were washed 1 time with 10 mLs of Isolation Bufferand placed into the magnet again for 2 minutes at room temperature, andthe bead-cleared buffer collected. The collected cells were centrifugedat 400×g for 5 minutes at RT. Supernatants were discarded, and the cellpellets were re-suspended in an arbitrary volume of RPMI Complete Mediadependent upon cell pellet size (2 to 35 mLs). Recovered CD3+ cells werethen counted on the ViCell and brought to a concentration of 1.2×10⁶cells/mL in RPMI Complete Media.

CD3 Confirmation Staining:

1×10⁶ recovered cells were stained with 5 μl anti-CD3 PE-Cy7 or 5 μlIgG1 Pe-Cy7 Isotype for 40 minutes at 4° C. in the dark. Cells were thenwashed twice in ice-cold PBS with 0.1% Tween20, centrifuging at 400×gfor 5 minutes at 4° C. Stained cells were re-suspended in 1%formaldehyde and incubated at 4° C. for 20 minutes in the dark. Cellswere then washed twice in ice-cold PBS with 0.1% Tween20, centrifugingat 400×g for 5 minutes at 4° C., and re-suspended in 275 μl of PBS with0.1% Tween20. Fixed cells were stored at 4° C. in the dark until FlowCytometry was performed to confirm the quality of the T cell isolation.

Activation of Isolated Human T Cells:

T75 flasks were coated with 4 mL of 1 μg/ml CD3/CD28 in PBS for 2 hoursat 37° C. Flasks were washed twice with 12 mL of PBS. 30×10⁶ cells in 25mLs of RPMI Complete Media were added per T75 flask. Cells wereincubated for 48 hours at 37° C., 5% CO₂ to allow activation to occur.Binding of Anti-ICOS (H2L5 hIgG4PE):

Binding of H2L5 hIgG4PE was assessed in both naive and activated CD3+ Tcells. An 8-point titration from 0.00128 to 100 μg/mL H2L5 hIgG4PE with5 fold dilutions was employed.

Either naive and/or activated CD3+ T cells were re-suspended in PBS with0.1% BSA (FACS Buffer) containing Human FcR Blocking Solution at 2×10⁶cells/mL (5 μl FcR Blocking Solution+950 μl FACS Buffer per 1 mL). At aconcentration of 2×10⁵ cells/well, 100 μl cells were placed into 2 mL 96well Assay Blocks and incubated at room temperature for 15 minutes.During the incubation, a titration of the binding antibodies, anti-ICOS(H2L5 hIgG4PE) or IgG4 Isotype Control antibody was prepared as a 2×concentration. Following the incubation of FcR block, 100 μl per well ofthe 2× concentrated binding antibodies were added to the 100 μl of Fcblocked T cells per well to achieve a final 1× concentration of antiICOS (H2L5 hIgG4PE) or IgG4 Isotype Control antibody from 0.00128 to 100μg/mL final concentration Cells were incubated with antibodies for 20minutes at room temperature. Following the binding incubation, cellswere washed twice in 1 mL of FACS buffer, centrifuging at 400×g for 5minutes at room temperature.

Staining of Naive or Activated T Cells Following Anti-ICOS (H2L5hIgG4PE) Binding:Cells were stained for flow cytometry with the following cocktails:

Staining Cocktail:

Antibody Vol. per well (μl) Vol. for 110 (μl) PE Mouse anti-Human CD4 5550 APC Mouse anti-Human CD8 5 550 FITC Goat anti-Human IgG Kappa 101100 Light Chain

Isotype Cocktail:

Antibody Vol. per well (μl) Vol. for 110 (μl) PE Mouse anti-Human CD4 5550 APC Mouse anti-Human CD8 5 550 FITC IgG1, Kappa Isotype Control 101100Naive or activated, Fc-blocked T cells were re-suspended in 80 μl ofFACS Buffer following the binding incubation with H2L5 hIgG4PE orcontrol antibodies. 20 μl per well of either the Staining or IsotypeCocktail was added per well. Cells were stained for 20 minutes at roomtemperature in the dark. Following the staining incubation, cells werewashed twice in 1 mL of FACS Buffer, centrifuging at 400×g for 5 minutesat room temperature.

Fixation of Stained Cells:

Cells were re-suspended in 500 μl of 1% formaldehyde (10 mLs of 16×concentrated Formaldehyde+150 mLs of 1×PBS) and incubated at roomtemperature for 20 minutes. Cells were then washed twice with 1 mL FACSbuffer, centrifuging at 400×g for 5 minutes at room temperature. Pelletswere then re-suspended in 265 μl of FACS Buffer and transferred to a 96well round bottom plate. Cells were stored at 4° C. in the dark untilanalysis by flow cytometry.

Flow Cytometry:

Flow cytometry was performed on either the FACS Fortessa X20 or the FACSCanto II using FACSDiva software (Version 8.0). Compensation wasperformed at the time of acquisition using single stained eBioscienceUltracomp beads and the compensation software in FACSDiva.

Data Analysis

Data acquisition and compensation were performed on BD FACS instruments,LSR Fortessa X-20 or FACS Canto II using BD Diva (ver. 8.0) software.Data analysis employed Flow Jo software (ver.10.0.8r1). Results arereported as both MFI (Median Fluorescence Intensity) and Percent ofcells positive for human IgG kappa light chain FITC staining out of thetotal live cells or appropriate parent population. EC50s were determinedusing Graphpad Prism 5 software (ver. 5.04) with non-linear regressionof transformed data (X=(log(X)) using a variable slope with 4 parameters(log(Agonist) vs. response—Variable slope).

Results

Isolation of T cells from fresh, whole human blood, using either RosetteSep CD3 Enrichment kit or Dynabead Untouched T cell isolation kits wasconfirmed by staining with anti-CD3 PeCy7. Donors ranged between 68% and97% positive for CD3 cells. Anti-CD3/anti-CD28 activated CD4+ and CD8+cell populations generated H2L5 hIgG4PE-concentration-dependent curveswhen assessed for anti-human IgG1 Kappa light chain FITC staining. H2L5hIgG4PE binding curves are presented as both percent anti-human IgG1Kappa light chain FITC positive and FITC median fluorescence intensity(MFI). T cells incubated with the IgG4 Isotype Control antibody did notproduce concentration dependent curves when assessed for anti-human IgG1Kappa light chain FITC staining. Naive CD4+ or CD8+ cells did notproduce full curves; however, there were concentration-dependentincreases observed from 0.1 μg/mL to 100 μg/mL.

The median (range) EC50 values were 1.04 μg/mL (0.628-1.31 μg/mL) forCD4+ FITC MFI and 0.652 μg/mL (0.27-0.74 μg/mL) for CD8+FITC MFI,respectively. The median (range) EC50 values were 0.834 μg/mL(0.45-0.965 μg/mL) for CD4+ Percent IgG Kappa Light Chain FITC Positiveand 0.583 μg/mL (0.371-1.23 μg/mL) for CD8+ Percent IgG Kappa LightChain FITC. (Table 10)

TABLE 10 Summary of 422 H2L5 hIgG4PE binding EC50 values to activatedhuman T cells Activated CD4 T cells Activated CD8 T cells PercentPercent Donor # MFI Positive MFI Positive 1124F36 0.628 0.45 0.564 0.6191149M52 1.31 0.882 0.74 0.547 1173F42 0.636 0.612 0.27 0.371 1123F591.04 0.853 Not Performed Not Performed 1141F45 1.27 0.965 Not PerformedNot Performed 2100M39 No Curve Fit 0.814 No Curve Fit 1.23  191F39 NoCurve Fit No Curve Fit No Curve Fit No Curve Fit 1155F49 No Curve Fit NoCurve Fit Not Performed Not Performed 1156F64 No Curve Fit No Curve FitNot Performed Not Performed Median 1.04 0.834 0.652 0.583 Mean 0.9770.763 0.525 0.692 Std. Dev. 0.331 0.193 0.237 0.374

Discussion

This study demonstrated that H2L5 hIgG4PE (anti-ICOS agonist antibody)bound to ICOS receptor on activated T cells from healthy human donors.H2L5 hIgG4PE binding to the cell surface of T cells was detected usingan antibody against human IgG kappa light chain labelled with FITC.

The successful isolation of CD3+ T cells was confirmed through flowcytometry with anti-CD3 Pe-Cy7 staining. Nine of ten donors resulted ingreater than 89% CD3+ T cells after isolation. However, Donor#2100M39was only 68.6% CD3+ following isolation. The cause of this decreasedpurity in Donor#2100M39's T cell isolation is unknown. EC50 valuesgenerated from gated CD4+ and CD8+ populations from Donor#2100M39 do notappear to be aberrant and were included in the summarized median values.

Binding EC50s were determined for H2L5 hIgG4PE in negatively isolatedhuman T cells. Binding curves were generated when the isolated T cellswere activated by 48 hours exposure to 1 μg/mL plate bound CD3/CD28antibodies. Both percent IgG kappa light chain FITC positive cells andFITC MFI data from CD4+ and CD8+ activated T cells were considered inthe statistical analyses. The median CD4+EC50 values were similar whencalculated as percent FITC positive cells or FITC MFI, 1.04 and 0.834μg/mL, respectively. The median CD8+EC50 values were also similar whencalculated as percent FITC positive cells or FITC MFI, 0.652 and 0.583μg/mL, respectively.

T cells incubated with the IgG4 PE isotype control did not result inconcentration-dependent increases in anti-human IgG kappa light chainFITC binding regardless of the analysis method employed, MFI or PercentPositive Cells.

Full curves could not be obtained from naive or unactivated, negativelyisolated T cells in the 0.00128 to 100 μg/mL H2L5 hIgG4PE range tested.However, a concentration-dependent increase in binding was observed indonors from 0.1 to 100 μg/mL H2L5 hIgG4PE. EC50s could not be calculatedas the curves from naive T cells were incomplete. The inability of H2L5hIgG4PE to bind at low concentrations to naive or unactivated cells wasexpected since ICOS is only weakly expressed on resting Th17, Tfollicular helper (TFH) and regulatory T (Treg) cells. TCR engagementand activation are required to induce ICOS expression. Thus, there waslikely very little ICOS receptor expressed on naive or unactivatedcells, and consequently minimal binding of H2L5 hIgG4PE.

Example 8 TK/PD Results from Cyno Dose-Range Finding (DRF) Study

To access the in vivo characteristics of H2L5 hIgG4PE in atarget-relevant species a dose-range finding study was performed incynomolgus monkeys. The study tested 3 dose levels (0.3, 3 and 30 mg/kg)in addition to a vehicle control cohort. It was a repeat dose with thesecond dose administered 14 days after the first. One male and onefemale were tested per cohort. H2L5 hIgG4PE exhibited a dose-dependentincrease in C_(max) (μg/mL) and AUC (μg·h/mL) across the 3 differentdoses which were tested. At all three dose levels, antibody was detectedin the plasma for two weeks following the first dose (FIG. 12A).Anti-H2L5 hIgG4PE antibodies were detected in 3 of the monkeys followinga single dose, both of the 0.3 mg/kg dosed animals as well as the femaledosed at 3 mg/kg. Anti-H2L5 hIgG4PE antibodies correlated with decreasedplasma concentrations following administration of the second dose inthese animals (FIG. 12B). Forty-eight hours after the second dose allanimals were sacrificed to collect tissue for analysis ofpharmacodynamic activity and histopathology analysis.

H2L5 hIgG4PE receptor occupancy (RO) was measured in CD4+ T cells fromthe spleens and axillary lymph nodes of all animals on study. Adose-dependent increase in H2L5 hIgG4PE binding was observed across thedose levels tested in both tissues (FIG. 13).

Receptor occupancy was also measured on CD4+ T cells from the peripheralblood of monkeys on study. Blood was drawn at 5 time points (Day 1(pre-dose), day 3, day 8, day 15 (pre-second dose) and day 17). Twodifferent measures were used in this assay to determine RO. The firstwas a “free-receptor” assay format in that, binding of the anti-ICOS mAbused for flow cytometry detection was determined in the presence orabsence H2L5 hIgG4PE which was shown to compete for ICOS binding.Therefore, the absence of anti-ICOS signal by FACS was a surrogate forH2L5 hIgG54PE occupied receptor and conversely, anti-ICOS positivityindicated “free receptor” which was unbound with H2L5 hIgG4PE. FIG. 14-Ashows that ICOS free receptor decreased in a dose-dependent, and timedependent manner. Two monkeys (250 and 300) demonstrated “free receptor”signals which could not be explained, and may be due to the productionof anti-H2L5 hIgG4PE antibodies in these monkeys. In addition, RO wasalso measured in peripheral blood CD4+ cells by the same assay used inspleen and lymph nodes described above. As would be expected the 0 mg/kgdose showed no RO by this read out (FIG. 14-B). Interestingly, somemonkeys at the 3.0 and 30 mg/kg dose levels showed a time dependentincrease in H2L5 hIgG4PE bound CD4+ cell numbers across the treatmenttime-course. In particular, animal 350 exhibited a (>5-fold) increase indrug-bound circulating CD4+ cells between days 3 and 17 (FIG. 14-B). Itis possible that this increase in CD4+ICOS+ cell number could be due toH2L5 hIgG4PE-induced proliferation of this population.

Example 9 H2L5 hIgG4PE Induces Intracellular Signalling Changes inResponse to Binding Experimental Preparation(s) Cell Lines

Ba/F3-ICOS cells were obtained from INSERM (Paris, France). Cells werecultured in the appropriate culture medium supplemented with 10% fetalbovine serum (FBS) (Sigma-Aldrich, St. Louis, Mo.), 10 ng/mL recombinantmurine IL-3 (R&D Systems, Minneapolis, Minn.), and 1 mg/mL Geneticin(ThermoFisher, Waltham, Mass.) at 37° C. in humidified incubators under5% CO₂.

Experimental Protocol(s) Intracellular Signalling Antibody Array

Protein lysates were assayed with the PathScan® Intracellular SignalingArray Kit (Cell Signaling Technologies) according to the manufacturer'sinstructions. Briefly, lysates from Ba/F3-ICOS cells treated withIgG4-PE (20 μg/mL) or H2L5 hIgG4PE (0.2, 2, or 20 μg/mL) for 1, 6, 24,and 48 hours were diluted to 1 μg/μL in Array Diluent Buffer andincubated overnight onto the antibody arrays at 4° C. Images of thearrays were captured using the Odyssey imaging software (LI-CORBiosciences, Lincoln, Nebr.).

Phospho-AKT Enzyme-Linked Immunosorbent Assay (ELISA)

Phosphorylation of AKT was measured using the Meso Scale Discovery (MSD)Phospho(Ser473)/Total Akt Whole Cell Lysate Kit and Phospho-Akt (Thr308)Whole Cell Lysate Kit according to the manufacturer's instructions.Cells were seeded at a cell density of 0.25×10⁶ cells/well into 96-wellU-bottom plates (BD Falcon) in the appropriate culture media (100μL/well). Cells were treated for 1, 2, 4, 6, 24, or 48 hours with eitherthe control antibody (IgG4 PE), anti-ICOS IgG1 Fc disabled antibody, orH2L5 hIgG4PE at 7 different concentrations using a 3-fold dilutionscheme (dose range: 20.0-0.03 μg/mL) in duplicate wells. For oneexperiment using the Phospho-AKT (Thr308) Whole Cell Lysate Kit, cellswere treated with one concentration of all three antibodies (10 μg/mL)in triplicate wells. The bottom row of each 96-well plate contained a nocells control (two blank duplicate wells) and cells that were leftuntreated with any antibody. Following treatment, cells were lysed with30 μL of ice cold lysis buffer containing protease and phosphataseinhibitors, incubated on ice for 30 minutes, and then 25 μL of lysatewas transferred to the ELISA plate to incubate overnight at 4° C.

Data Analysis Densitometry Analysis of Intracellular Signalling AntibodyArray

Densitometry analysis was performed to calculate the integratedintensity levels of the spots across the antibody array. The intensitiesfor each spot were normalized to the average of the positive controls onthe array (formula=sample well/average of positive controls) and graphedusing GraphPad Prism 6.0 (La Jolla, Calif.).

Analysis of MSD ELISA Data

The percent phosphoprotein was calculated for each well using thefollowing calculation: % Phosphoprotein=((2×Phospho-signal)/(Phospho-signal+Total protein signal))×100. This valuewas then normalized to the untreated cells value at each time point andgraphed as “% control” in Microsoft Excel 2007.

Results

Prior studies demonstrated that H2L5 hIgG4PE treatment increasedphospho-AKT (S473) levels in Ba/F3-ICOS cells with the maximum responseobserved between 30-40 minutes after antibody exposure. Here,phospho-AKT levels were measured at later time points to see ifincreased phosphorylation levels persisted after several days.Additionally, the regulation of other intracellular signalling events byICOS activation was assessed. In Ba/F3-ICOS cells, phospho-AKT (S473)levels were increased with H2L5 HIGG4PE treatment compared to IgG4-PEisotype control antibody-treated cells after one and six hours oftreatment, but this effect was lost after 24 hours (FIG. 15).Interestingly, a similar effect was observed when cells were treatedwith an anti-ICOS antibody where the Fc region of the antibody isdisabled. Increased levels of phospho-AKT (T308) were also observed inH2L5 hIgG4PE and anti-ICOS IgG1 Fc disabled antibody-treated cellscompared to IgG4-PE (FIG. 15) isotype control antibody-treated cellsafter one hour of treatment and persisted up to 48 hours which was thelast time point measured. Two other phospho-proteins downstream of AKT,Glycogen Synthase Kinase 3 Alpha (GSK3α) and ribosomal protein S6, werealso modestly increased upon ICOS activation, but the effects were notas robust at those seen with phospho-AKT. Protein lysates were alsoanalyzed using an antibody array that measures the phosphorylation orcleavage of 18 proteins involved in intracellular signalling. Using thisapproach, only three proteins showed slight increases in phosphorylationupon ICOS activation: phospho-AKT (S473), phospho S6 (S235/236), andphospho-SAPK/JNK (T183/Y185).

To measure changes to AKT phosphorylation using an assay format thatwould allow for direct quantitation, Ba/F3-ICOS cells were treated witha dose range of control antibody (IgG4 PE), anti-ICOS IgG1 Fc disabledantibody, or H2L5 hIgG4PE over time and monitored by ELISA. Increasedphospho-AKT (S473) levels were both dose-dependent and time-dependent inanti-ICOS IgG1 Fc disabled antibody-treated or H2L5 hIgG4PE-treatedcells. As previously observed, maximal phospho-AKT (S473) activationoccurred after 1 hour of treatment. The phospho signal slightlydecreased after 2 hours and persisted up to 6 hours but was eventuallylost after 24 hours. An ELISA that measures phospho-AKT (T308) levelswas also tested here but no reproducible activation could be observedwith this ELISA kit.

Discussion

The AKT signalling cascade can be activated by receptor tyrosinekinases, integrins, B and T cell receptors, cytokine receptors,G-protein-coupled receptors and other stimuli that induce production ofphosphatidylinositol (3,4,5) trisphosphates (PIP3) by PI3K [Carnero,2008]. These lipids serve as plasma membrane docking sites for Akt andits upstream activator PDK1. At the membrane, PDK1 phosphorylates AKT atThr308 leading to partial activation of Akt [Alessi, 1996].Phosphorylation of Akt at Ser473 by mTORC2 stimulates full enzymaticactivity [Sarbassov, 2005].

ICOS plays a key role in the function of activated effector andregulatory CD4+ T cells by promoting T cell survival, proliferation andmemory. Due to its role in sustaining T-cell activation and effectorfunctions, targeting ICOS with an agonist antibody could be a plausibleapproach to enhance antitumor immunity. In this study, we observed thatactivation of ICOS by H2L5 hIgG4PE caused changes to AKT phosphorylationin Ba/F3-ICOS cells. Subsequently, proteins downstream of AKT, such asGSK3α (a direct substrate of AKT) and ribosomal protein S6 were alsophosphorylated. This data is consistent with work performed recentlywith this model system, and is in-line with data published externally[Fos, 2008].

Example 10 Functional Effects of Soluble H2L5 hIgG4PE Alone and inCombination with Anti-PD1 and Anti-CTLA-4 Antibody in Human PBMC AssayExperimental Preparation(s) Isolation of Primary Human PBMC

Fresh blood was obtained from GSK Health Center blood donors and wasdiluted 1:1 with phenol red free-10% RPMI1640 media. Diluted blood waslayered on top of the density medium in a Uni-Sep Max 50 ml conical tubeand centrifuge at 400×g for 20 minutes at room temperature with BREAKOFF. The resulted white mononuclear layer (buffy coat) was carefullyextracted into a new 50 mL conical tube through a 100 μM cell strainer.An equal volume of Phenol red free-10% RPMI1640 media was added to thebuffy coat and centrifuged at 300×g for 10 minutes at room temperature.The cell pellet was resuspended in 10 ml of red blood cell lysissolution (Sigma Aldrich) and incubated for 5 minutes at roomtemperature. Cells were washed once with media and centrifuged aspreviously described. Volume was brought to 40 ml with Phenol redfree-10% RPMI1640 media and cells were counted using Vicell cell counterand viability analyzer (Beckman Coulter).

Induction of Monocyte-Derived Immature Dendritic Cells (iDC)

Human monocytes were isolated using the plastic adherence method.Briefly, 20 million freshly isolated PBMC were cultured in a T-75 tissueculture flask in AIM-V media (Thermo Fisher) for 3 hours. Cells that donot bind to plastic were washed off. The adherent monocytes werecultured in a 37° C. 5% CO₂ incubator in AIM-V media supplemented with1000 U/ml of human GM-CSF (Calt#300-03, PeproTech) and 500 U/ml of humanIL-4 (cat#200-04). After 7-10 days, the iDC cells were collected forco-culturing with T cells from a different donor in the allogeneic MixedLymphocyte Reaction assays.

Isolation of Primary Human T Cells Directly from Blood

Human T cells were isolated directly from fresh human blood using ahuman T cell enrichment cocktail (Stem Cell Technologies). TheRosetteSep Human T Cell Enrichment Cocktail (50 μL/mL) was added towhole blood and mixed well. After 20 minutes of incubation at roomtemperature, an equal volume of PBS+2% FBS was added with gentle mixing.The diluted sample was layered on top of the density medium andcentrifuged for 20 minutes at 1200×g at room temperature with the brakeoff. The enriched cells from the density medium: plasma interface werecarefully poured into a new conical tube. Next, the red blood cells werelysed with Red Blood Cell Lying Buffer (Sigma Aldrich) and the enrichedcells were washed with PBS+2% FBS twice. The T cells were thenresuspended in 40 ml of PBS+2% FBS and counted with a Vi-Cell cellcounter.

Experimental Protocols Human PBMC Pre-Stimulation Assay

Freshly isolated human PBMCs were pre-stimulated with CD3/CD28 T cellexpander DynaBeads at a bead to cell ratio of 1:20 in a T-75 tissueculture flask in AIM-V medium supplemented with 100 ng/ml of MCSF and100 IU/ml of IL-2 (PeproTech) at 37° C. After 48 hours, thepre-stimulation beads were magnetically removed and cells were washed,counted and re-stimulated with anti-CD3 DynaBeads and therapeuticantibodies in AIM-V medium supplemented with 100 IU/ml of IL-2(PeproTech) in 96-well non-tissue culture treated round bottom plate.The seeding density was 100 k cells per 100 μl of medium per well. Afterincubating at 37° C. for 3.5 days, cell culture supernatants werecollected for multiplex cytokine measurement by MSD.

Human MLR Activation Assay

Monocyte-derived iDCs from a healthy human volunteer were mixed at a1:10 ratio (iDC:T) with freshly isolated human T cells from a differentdonor and pre-incubated at 37° C. in AIM-V media in the presence of 0.02μg/ml of a CEFT peptide mixture for 24 hours. Different groups oftreatment antibodies were added directly to the wells, mixed and furtherincubated for an additional 4 days. Cell culture supernatants werecollected for multiplex cytokine measurement by MSD analysis.

MSD Cytokine Analysis

IFN-γ, IL-10, IL-2 and TNF-α cytokine levels in the tissue culturesupernatant were determined using MSD human V-Plex customized kits.Samples were first diluted 1:200 in Diluent 2. Calibrators were alsoprepared in Diluent 2 following the manufacturer's recommendations.Diluted samples and calibrators were added to black MSD plates whichwere subsequently sealed with an adhesive plate seal and incubated atroom temperature with shaking for 2 hours. After adding 25 μL of thedetection antibody solution, which was freshly prepared in Diluent 2 toeach well, the plate was re-sealed and incubated at room temperaturewith shaking for another 2 hours. The plates were washed 3 times with150 μL/well of PBS plus 0.05% Tween-20 before adding 150 μl/well offreshly diluted 2× read buffer and immediately read on a MESO QuickPlexreader. Data were analyzed using MSD Workbench software.

Data Analysis MSD Data Analysis

MSD data was analyzed with Discovery Workbench software (MSD, version4.0.9). Calibrators in the manufacturer's kit were included on each MSDplate to generate plate specific standard curves with R² value over 0.99in all cases. The amounts of cytokine detected were back calculatedbased on the standard curve and the mean and standard deviation fromthree biological replicates were used to generate the graphs.

Statistical Analysis

One-way ANOVA was performed on log-transformed, fold-change data overeach treatment antibody's own isotype control. Dunnett's MultipleComparison Test was performed to compare both mono-therapies vs.combination across different donors. P<0.05 was considered asstatistical significant.

Results

PBMC pre-stimulation assay development and test for combinatorialactivity of H2L5 hIgG4PE with ipilimumab and pembrolizumab.

In order to determine the optimal conditions for pre-stimulation, humananti-CD3 Dynabeads and anti-CD3/CD28 Dynabeads (Thermo Fisher) weretested at different bead to cell ratios. After 48 hour pre-stimulation,cells were harvested and beads were magnetically removed prior tostimulation with anti-CD3 Dynabeads (bead to cell ratio=1:1) togetherwith anti-ICOS antibody alone or in combination with anti-CTLA-4 oranti-PD1. H2L5 hIgG4PE single agent treatment resulted in induction ofIFN-γ as compared to isotype control in all pre-stimulation conditionstested. The magnitude of IFN-γ induced by H2L5 hIgG4PE was inverselycorrelated with the strength of the pre-stimulation. The combination ofH2L5 hIgG4PE together with ipilimumab demonstrated enhanced cytokineproduction as compared to either H2L5 HIGG4PE or ipilimumab alone inPBMCs that were weakly pre-stimulated. The combination effect was lostunder plate-bound anti-CD3/anti-CD28 pre-stimulation conditions, whichis considered a stronger pre-stimulation condition. Based upon theseresults, the pre-stimulation condition using anti-CD3/anti-CD28 beads ata bead to cell ratio of 1:20 was chosen for all future PBMC assays.Results from four individual donors are summarized for anti-CTLA-4combination in FIG. 16 and combination with anti-PD-1 in FIG. 17.

H2L5 hIgG4PE Results in Dose-Dependent Cytokine Induction in a PBMCPre-Stimulation Assay

The dose-dependent activity of H2L5 hIgG4PE was evaluated in human PBMCspre-stimulated with anti-CD3/anti-CD28 beads at a pre-determined bead tocell ratio of 1:20. The anti-RSV IgG4PE and anti-ICOS 422.2 IgG1 FcDisabled were included as controls. Eight concentrations of H2L5 HIGG4PEwere tested (100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 μg/ml). IFN-γ, IL-10and TNF-α were evaluated by MSD in the tissue culture supernatants ofPBMC samples. H2L5 hIgG4PE, but not isotype control IgG4 or Fc-Disabled422.2, induced IFN-γ, IL-10 and TNF-α production in a dose-dependentmanner. These results were used to determine the concentration of H2L5hIgG4PE to be used in combination studies.

Human MLR Assay Development

In an effort to optimize a human MLR assay, in addition to co-culture ofhuman T cells and monocyte-derived immature DCs from a different donor,anti-CD3 beads were also added into the wells to provide a basal TCRstimuli to help prime the cells. Results demonstrated that anti-CD3beads greatly increased the range of IFN-γ induction. Althoughipilimumab alone can induce IFN-γ production in the absence of anti-CD3beads, H2L5 hIgG4PE alone or the H2L5 HIGG4PE/ipilimumab combinationonly showed enhanced IFN-γ production over corresponding controls in thepresence of anti-CD3 beads.

Combinatorial Activity of H2L5 HIGG4PE and Ipilimumab in a Human MLRAssay

The immunostimulatory activity of H2L5 hIgG4PE alone or in combinationwith ipilimumab was tested in an allogeneic human MLR assay in which Tcells that were pre-incubated with monocyte-derived immature DCs from anunmatched donor in the presence of 0.02 μg/ml CEFT peptides for 1 day.The H2L5 hIgG4PE/ipilimumab combination resulted in a significantenhancement in IFN-γ production as compared to either agent alone.Results were consistent across three donor pairs tested; however, modestvariability was observed between donors (FIG. 18).

Combinatorial Activity of H2L5 hIgG4PE and Pembrolizumab in a Human MLRAssay

The combination of H2L5 hIgG4PE and pembrolizumab was also tested in thehuman allogeneic MLR assay described above. H2L5 hIg G4PE was testedalone and in combination with pembrolizumab at 10 μg/ml. The combinationof H2L5 hIg G4PE and pembrolizumab resulted in increased IFN-γ ascompared to either agent alone. However, statistical significance wasnot reached due to high donor variability and significant activity ofsingle agent anti-PD-1 treatment in some donors (FIG. 19).

Discussion

ICOS is a costimulatory receptor that is weakly expressed on naive Tcells and quickly upregulated in activated CD4+ and CD8+ T cells. Theligand for ICOS is ICOS-L (B7h, B7RP-1, CD275), which is expressed byprofessional APCs and by peripheral epithelial and endothelial cellsfollowing TNF-α stimulation. The ICOS:ICOS-L pathway provides a keycostimulatory signal for T-cell proliferation and function. Due to itsrole in sustaining T-cell activation and effector functions, targetingICOS by agonist antibodies could be a plausible approach to enhanceanti-tumor immunity.

Studies have shown an increase the frequency of ICOS″ CD4+ effector Tcells after CTLA-4 blockade by ipilimumab in several cancer models. Inaddition, upon CTLA-4 blockade, this cell population produced greaterlevels of INF-γ than ICOS^(lo) CD4+ T cells. In fact, the increase inthe frequency of ICOS+CD4 T cells has been identified as apharmacodynamic biomarker of ipilimumab treatment in cancer patients.Studies, in wild-type C57BL/6 mice, demonstrated 80 to 90% tumorrejection follow CTLA-4 blockade therapy; however, in ICOS or ICOSLknockout mice the efficacy was decreased to less than 50%. The importantrole played by ICOS in the effectiveness of CLTA-4 blockade suggeststhat stimulating the ICOS pathway during anti-CTLA-4 therapy mightincrease therapeutic efficacy. Therefore, we set out to evaluate thecombination activity of H2L5 hIgG4PE and ipilimumab.

Programmed cell death-1 (PD-1) was reported in 2000 to be another immunecheckpoint molecule. The expression of PD-L1 (B7-H1), which is one ofthe ligands of PD-1, can be found on many cell types including T cells,epithelial cells, endothelial cells, and tumor cells. Antibodiestargeting the PD-1/PD-L1 axis have also shown clinical responses inmultiple tumor types. The FDA recently approved pembrolizumab andnivolumab as second generation of the immune checkpoint blockers for thetreatment of cancer. Merck's pembrolizumab was shown to lead to responserates of ˜37 to 38% in patients with advanced melanoma, with asubsequent study reporting an overall response rate of 26% in patientswho had progressive disease after prior ipilimumab treatment. Nivolumab,the anti-PD-1 antibody from BMS, also showed clinical benefit inpatients with metastatic melanoma with a response rate of 40% and anoverall survival rate of 72.9% at 1 year. In addition, nivolumab wasalso FDA-approved for advanced or metastatic non-small cell lung cancer.As the PD-1 checkpoint blockade antibodies become the dominant cancerimmune therapy in the clinic, it will be important to evaluate H2L5hIgG4PE in combination with an anti-PD-1 antibody for their combinedanti-tumor activity.

Previously, a PBMC activation assay was developed and used to evaluatethe T cell stimulation activity of a panel of anti-ICOS agonistantibodies. The data generated from those studies supported thecandidate selection of clone 422.2 with an IgG4PE isotype as H2L5hIgG4PE. In the previous assay, PBMC cells were pre-stimulated withplate bound anti-CD3 antibody at 1 μg/ml and anti-CD28 antibody at 3μg/ml for 48 hours before they were harvested and re-stimulated withanti-CD3 and soluble ICOS antibodies that were being investigated. H2L5hIgG4PE was shown to induce IFN-γ production in a dose-dependent manner.In order to determine the optimal conditions for pre-stimulation, humananti-CD3 Dynabeads and anti-CD3/CD28 Dynabeads (Thermo Fisher) weretested at different bead to cell ratios. Stimulation by beads isconsidered to be more physiological and the strength of the stimulationcan be controlled more easily by constructing different bead to cellratios. After 48 hours of pre-stimulation, cells were harvested andbeads were magnetically removed prior to stimulation with anti-CD3Dynabeads (bead to cell ratio=1:1) together with anti-ICOS antibodyalone or in combination with anti-CTLA-4. The results showed that H2L5hIgG4PE single agent treatment resulted in IFN-γ induction relative toisotype control in all pre-stimulation conditions tested. The magnitudeof IFN-γ induced by H2L5 hIgG4PE was inversely correlated with thestrength of the pre-stimulation. The combination of H2L5 hIgG4PEtogether with ipilimumab demonstrated enhanced cytokine production ascompared to either H2L5 hIgG4PE or ipilimumab alone in PBMCs that wereweakly pre-stimulated. The combination effect was lost under plate-boundanti-CD3/anti-CD28 pre-stimulation conditions, which is considered astronger pre-stimulation condition. Based upon these results, thepre-stimulation condition using anti-CD3/anti-CD28 at a bead to cellratio of 1:20 was chosen for all the future PBMC assays. H2L5 hIgG4PEand ipilimumab combination demonstrated a statistically significantincrease in IFN-γ production as compared to either antibody treatmentalone.

In the assay optimization effort, with an anti-CD3/anti-CD28 stimulationbead to cell ratio fixed at 1:20, the anti-CD3 beads used during there-stimulation step were titrated down from bead to cell ratios of 1:1to 1:3 and 1:10. The results showed that the lower the re-stimulationstrength yielded lower the IFN-γ induction by H2L5 hIgG4PE. Thecombination effect by H2L5 hIgG4PE and ipilimumab was totally lost underre-stimulation at a bead to cell ratio of 1:3 and 1:10. Therefore, there-stimulation anti-CD3 bead to cell ratio of 1:1 was kept for allfuture experiments.

With the pre-stimulation and re-stimulation conditions optimized, thisassay was used to evaluate the dose response of H2L5 hIgG4PE. A total of8 antibody concentrations were tested, which were 100, 30, 10, 3, 1,0.3, 0.1 and 0.03 μg/ml. The anti-RSV IgG4PE and anti-ICOS 422.2 IgG1 FcDisabled, the Fc Disabled version of H2L5 hIgG4PE, were used ascontrols. Results showed that H2L5 hIgG4PE, but not isotype control IgG4or Fc-Disabled 422.2, induced IFN-γ, IL-10 and TNF-α production in adose-dependent manner. It is interesting that the Fc Disabled version ofH2L5 hIg G4PE exhibited a limited cytokine induction response, indictingthe Fc receptor engagement is crucial for the T cell agonizing functionof H2L5 hIg G4PE. These results were also used to determine the dose ofH2L5 hIg G4PE for combination studies.

A mixed lymphocytes reaction (MLR) assay was also developed to evaluatethe combination effect of H2L5 hIg G4PE and checkpoint blockingantibodies. MLR assay is an ex vivo cellular immune assay in whichprimary monocyte-derived immature dendritic cells (iDCs) were mixed withT cells isolated from a different donor. The mismatch of majorhistocompatibility complex (MHC) molecules on the surface of iDC cellscan initiate T cell stimulation in an allogeneic setting. In the clinic,the MLR assay is well-known for identifying the compatibility of tissuetransplants between donors and recipients.

In order to develop the MLR assay, fresh human monocytes were culturedin medium supplemented with human recombinant GM-CSF and IL-4 for a weekto induce an immature DC phenotype. Then fresh human T cells from adifferent donor were isolated and mixed with the iDC cells at a 10:1ratio (T:iDC). H2L5 hIg G4PE and ipilimumab mono-therapy orcombinational treatments were added to the T cell/iDC co-culture in thepresence or absence of anti-CD3 beads. The purpose of the anti-CD3 beadswas to provide a basal TCR stimulus to help prime the T cells. Resultsshowed anti-CD3 beads greatly increased the range of IFN-γ induction inthe assay. Although ipilimumab alone can induce IFN-γ production in theabsence of anti-CD3 beads, H2L5 hIgG4PE alone or the H2L5hIgG4PE/ipilimumab combination showed enhanced IFN-γ production overcorresponding controls in the presence of anti-CD3 beads. This resultsuggests that, in this assay, the TCR stimulus by DC cells alone may notbe sufficient to induce ICOS expression on the surface of resting Tcells that were freshly isolated from PBMCs. In order to improve thesituation, a 24 hour iDC and T cells pre-incubation step was addedbefore the addition of therapeutic antibodies. The CEFT peptide mix wasalso added into the assay procedure to better prime the T cells and toelicit an antigen-specific response. The CEFT peptide pool consists of27 peptides selected from defined HLA class I and II-restricted T-cellepitopes from human Cytomegalovirus (HHV-5; CMV), Epstein-Barr virus(HHV-4; EBV), Influenza A and Clostridium tetani. Considering the highvaccination frequency against Influenza and Clostridium tetani and thehigh prevalence of CMV and EBV in the general population, recall antigenresponses were expected for a majority of the human samples. The resultsshowed that increased IFN-γ production was observed when T cells werepre-incubated with iDC cells for 24 hours, and the IFN-γ productionfurther increased when CEFT peptides were added to the co-culturesystem. The immunostimulatory activity of H2L5 hIgG4PE alone or incombination with ipilimumab was tested in the allogeneic human MLR assayin which T cells that were pre-incubated with monocyte-derived immatureDCs from an unmatched donor in the presence of 0.02 μg/ml CEFT peptidesfor 1 day. The H2L5 hIgG4PE/ipilimumab combination resulted in asignificant enhancement in IFN-γ production as compared to either agentalone. The results were consistent across three donor pairs tested;however, modest variability was observed between donors.

Similarly, the combination of H2L5 hIgG4PE and pembrolizumab was alsotested in the human allogeneic MLR assay described above. H2L5 hIgG4PEwas tested alone and in combination with pembrolizumab at 10 μg/ml. Thecombination of H2L5 hIgG4PE and pembrolizumab resulted in increasedIFN-γ as compared to either agent alone. However, statisticalsignificance was not reached due to high donor variability andsignificant activity of single agent anti-PD-1 treatment in some donors.

In summary, these studies demonstrated the superior combination activityof H2L5 hIgG4PE with two FDA-approved check point inhibitors, ipilimumaband pembrolizumab, when compared to mono-therapies in two human immunecell based assays. In the studies reported here, H2L5 hIgG4PE was shownto promote T cell activation and T_(H)1 skewing (e.g. IFN-γ production)that is characteristic of productive anti-tumor immune responses.

Example 11 Functional Activity of H2L5 hIgG4PE Alone and in Combinationwith Anti-PD1 and Anti-CTLA-4 Antibodies In Vivo Human PBMC Mouse TumorModel Methods Experimental Preparations

All procedures on animals were reviewed and approved by the GSKInstitutional Animal Care and Use Committee prior to initiation of thestudies protocol.

Preparation of Cell Lines:

A2058 were propagated according to ATCC protocol.

Materials:

-   -   A2058 human melanoma cell line: ATCC, Cat# CRL-11147,        lot#59349362    -   DPBS: ATCC, Cat #30-2200, Lot#63357436    -   Dulbecco's Modified Eagle's Medium: ATCC, Cat #30-2002,        Lot#62596471 Expiration: October-2015    -   Fetal Bovine Serum: Sigma-Aldrich, Cat#12176c-1000 ml, lot        #13G180R0H1, Expiration: July-2018    -   0.25% (w/v) Trypsin—0.53 mM EDTA: ATCC, Cat #30-2102,        Lot#62420300    -   Antibiotic-Antimycotic (100×): Life Technologies, Cat#15240-062    -   T175 cell culture flask: Greiner bio-one, Cat#661175    -   T75 cell culture flask: Greiner bio-one, Cat#658175

Medium:

-   -   A2058 complete growth medium: Dulbecco's Modified Eagle's        Medium+10% FBS. Culture conditions: Atmosphere: Air, 95%; 5%        carbon dioxide (CO2); Temperature: 37° C.    -   Upon receipt of the cells:    -   Pre-warm complete medium at 37° C.    -   Thaw the cells quickly in 37° C. water bath. Wipe the tube with        70% ethanol and transfer cells to 15 ml tube filled with        prewarmed complete medium.    -   Centrifuge at 1200 rpm for 5 minutes to collect the cell pellet.    -   Add the cells back to T75 flask filled with prewarmed complete        medium and incubate at 37° C.

Subculture of the Cells:

-   -   Volumes are given for a 75 cm² flask (For T175 cm² flask, adjust        the amount of dissociation and culture medium needed        proportionally).    -   Remove and discard culture medium.    -   Briefly rinse the cell layer with DPBS to remove all traces of        serum that contains trypsin inhibitor.    -   Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe        cells under an inverted microscope until cell layer is dispersed        (2-3 minutes).    -   Note: To avoid clumping do not agitate the cells by hitting or        shaking the flask while waiting for the cells to detach. Cells        that are difficult to detach may be placed at 37° C. to        facilitate dispersal.    -   Add 10 mL of complete growth medium and aspirate cells by gently        pipetting.    -   Centrifuge at 1200 rpm for 5 minutes to collect the cell pellet,        add 10 ml of complete growth medium    -   Add appropriate aliquots of the cell suspension to new culture        vessels. Incubate cultures at 37° C.    -   Medium Renewal: Every 2 to 3 days

Preparation of Tumor Cells for Mice Inoculation:

-   -   Wash cells with 1×DPBS, add 3 ml 1× Trypsin for 2-3 minutes.    -   Add complete growth media and collect the cell suspension in        sterile conical centrifuge tube in the tissue culture hood.    -   Centrifuge the cells at 1200 rpm for 5 minutes to obtain cell        pellet.    -   Wash cells with 1×DPBS solution, Centrifuge at 1200 rpm for 5        minutes to obtain cell pellet. Repeat the washing 2 times.    -   Count the cells by hemocytometer for cell number and viability.    -   Resuspend the cells in ice-cold PBS at concentrations for In        Vivo inoculation (A2058, 2.5e7/ml, 2.5e6/100 μl/mouse).

Tumor Cell Line Inoculation to NSG Mice Materials:

-   -   Mouse: NOD.Cg-Prkdcscid Il2rgtm1Wj1/SzJ. The Jackson Laboratory        Stock: 005557 Female Age: 6 weeks    -   1 mL Tuberculin Syringes with Attached Needle 25 G 5/8: Becton        Dickinson, Cat#305554    -   PDI™ Alcohol Prep Pads: Professional Disposables, Cat# B339    -   PDI™ Povidone-Iodine Prep: Pad Professional Disposables,    -   Cat# B40600Medium; 100/box, 10 boxes/Cs.    -   Preparation of mice    -   Mice should be 6 weeks old.    -   Allow 3-5 days acclimatization period after mice have arrived.    -   Shave the mice on right hind flank

Preparation of the Injection

-   -   Clean and sterilize the inoculation area of the mice with iodine        followed by ethanol pad    -   Use 1-cc syringe and a 25-gauge needle    -   Pull out the plunger, mix cells and add 100 μl of cells to the        back of syringe, carefully insert the plunger.    -   Inject cells subcutaneously (s.c.) into the right hind flank of        the mouse.    -   Tumor Growth Assessment    -   To measure a tumor, wet fur with 70% ethanol to make it easier        to find tumor margins. Measure tumor size and body weight every        2-3 days.    -   Tumor size is measured with a digital caliper, and the volume is        determined as follow: Tumor volume (mm3)=(length)×(width)²/2    -   Human PBMC Intravenous Administration    -   Human PBMC administration can start 1 week after when the tumors        have reached an average volume of approximately 100 mm3.

Materials:

-   -   Fresh Human PBMC: Allcells, cat#C-PB102-3B2    -   1 mL Tuberculin Syringes with Attached Needle 25 G 5/8: Becton        Dickinson, Cat#305554    -   PDI™ Alcohol Prep Pads: Professional Disposables, Cat# B339    -   PDI™ Povidone-Iodine Prep Pad Professional Disposables, Cat        B40600    -   Gauze sponges: Covidien, cat#441211    -   Mouse Tail Illuminator Restrainer: Braintree scientific, cat#MTI        STD    -   PBMC preparation    -   Fresh human PBMC are purchased from Allcells by overnight        shipment.    -   Centrifuge the cells at 1400 rpm for 5 minutes to obtain cell        pellet.    -   Wash cells with 1×DPBS solution, Centrifuge at 1400 rpm for 5        minutes to obtain cell pellet.    -   Resuspend the cells in ice-cold PBS at concentrations for In        Vivo injection (20e7/ml).    -   Use 1-cc syringe and a 25-gauge needle.    -   Pull out the plunger, mix cells and add 100 μl of cells to the        back of syringe, carefully insert the plunger.    -   Keep the cells on ice.    -   Tail Vein injection    -   Warm the mice with an incandescent lamp for 5 minutes    -   Restrain the mice with tail illuminator restrainer.    -   Rotate the tail slightly to visualize vein.    -   Clean and sterilize injection site with iodine followed by        ethanol pad    -   Insert needle into the vein at a slight angle and inject the        cells.    -   Remove the needle and apply gentle compression with Gauze        sponges until bleeding has stopped.    -   Return animals to their cage and observe for 5-10 minutes to        make sure that bleeding has not resumed.

Therapeutic Antibody Administration

-   -   1-3 days post human PBMC injection, mice are administrated with        antibodies by Intraperitoneal injection.

Materials:

-   -   Fully human IgG1 isotype control: Eureka therapeutics,        cat#ET-901 (preclinical grade) Lot#15-726 Expiration: February        2017    -   Ipilimumab (Yervoy): Bristol-Myers Squibb NDC 0003-2327-11,        lot#921873 Expiration: April 2015; lot#4H69490, Expiration: May        2016    -   Fully human IgG4 isotype control: Eureka therapeutics,        cat#ET-904 (preclinical grade) Lot#15-726 Expiration: February        2017    -   Anti human ICOS H2L5 hIgG4PE    -   Pembrolizumab (Keytruda): Merck, NDC 0006-3026-02, lot# L010592,        Expiration: Apr. 26 2016    -   Intraperitoneal injection:    -   Draw up, into the syringe and needle, 100 μl of to be        administered.    -   Line the bevel of the needle with the numbers on the syringe.    -   Sufficiently restrain the animal with your non-dominant hand.    -   Entry point for the needle: Draw an imaginary line across the        abdomen just above the knees, the needle will be inserted along        this line on the animal's right side and close to the midline.        As this is a female, you can see that the point of entry is        cranial to and slightly medial of the last nipple.    -   Tilt the mouse with its head slightly toward the ground so that        its head is lower than its hind end.    -   Insert the needle into the abdomen at about a 30-degree angle.    -   The shaft of the needle should enter to a depth of about half a        centimeter.    -   After injection, withdraw the needle and return the mouse to its        cage.    -   Blood and tumor sampling    -   Materials:    -   Microvette CB300 (Serum): Braintree Scientific, Cat# MV-CB300        16440    -   Microvette CB300 (Hematology/Potassium EDTA): Braintree        Scientific, Cat# MV-CB300 16444    -   Blood:    -   Mice were tail vein bled once a week.    -   30 μl of blood was collected in Microvette CB300        (Hematology/Potassium EDTA) for flow cytometry analysis.    -   Another 30 μl of blood was collected in serum Microvette CB300        and incubated for 2 hours at room temperature to allow clotting,        followed by centrifugation at 2000×g in order to collect serum.        Serum was stored at −20 until further analysis.

Tumor:

-   -   Mice were euthanized when tumor size reached 2000 mm³. Tumors        were collected and processed in the following procedure.

Experimental Design

All studies were prepared according to procedures listed above.H2L5 hIgG4PE Dose Response

This study was designed to determine dose-dependent activity of H2L5hIgG4PE in human PBMC engrafted NSG mice implanted with A2058 melanomatumors. Nine groups with 10 mice per group and 1 control group (Tumoronly no PBMC) with 7 mice were assigned into each study. A summary ofthe treatment regimen for dose response using human PBMC from donor#7129 is present in Table 11. H2L5 hIgG4PE was dosed at 0.04, 0.4, 1.2and 4 mg/kg. Ipilimumab was dosed at 3 mg/kg and an Fc-Disabled variantof the anti-ICOS agonist was tested at 1 mg/kg. Test groups wereevaluated relative to the vehicle and matched isotype control groups.Survivability analysis concluded on day 49 at termination of the study.

TABLE 11 Summary of Treatment Regimen for H2L5 hIgG4PE Dose Response inMice # mice/ Groups Treatment 1 Treatment 2 group Dosing 1 Tumor +Vehicle 10 Twice weekly for huPBMC (donor 3 weeks #7129) 2 Tumor + humanIgG1 Isotype 10 Twice weekly for huPBMC (3 mg/kg) 3 weeks (donor #7129)3 Tumor + Ipilimumab (3 mg/kg) 10 Twice weekly for huPBMC 3 weeks (donor#7129) 4 Tumor + human IgG4 (4 mg/kg) 10 Twice weekly for huPBMC 3 weeks(donor #7129) 5 Tumor + H2L5 hIgG4PE (0.04 mg/kg) 10 Twice weekly forhuPBMC 3 weeks (donor #7129) 6 Tumor + H2L5 hIgG4PE (0.4 mg/kg) 10 Twiceweekly for huPBMC 3 weeks (donor #7129) 7 Tumor + H2L5 hIgG4PE (1.2mg/kg) 10 Twice weekly for huPBMC 3 weeks (donor #7129) 8 Tumor + H2L5hIgG4PE (4 mg/kg) 10 Twice weekly for huPBMC 3 weeks (donor #7129) 9Tumor + ICOS-Fc-disabled 10 Twice weekly for huPBMC (1 mg/kg) 3 weeks(donor #7129) 10 Tumor (no Untreated 7 Twice weekly for PBMC) 3 weeks(donor #7129)Efficacy and Pharmacodynamic (PD) Activity Study with H2L5 hIgG4PE inCombination with Ipilimumab and Pembrolizumab

Study Objectives:

To evaluate the anti-tumor activity of H2L5 hIgG4PE monotherapy dosed at0.04 mg/kg and 0.4 mg/kg.

To evaluate the anti-tumor activity of H2L5 hIgG4PE dosed in combinationwith ipilimumab or pembrolizumab with matched isotype controls.

Collection of tissue for future pharmacodynamic activity study of H2L5hIgG4PE. A total of 22 treatment groups with 10 mice per group wereassigned to this study. Groups 1-16 were the efficacy cohorts and 17-22were pharmacodynamic activity cohorts.

For combination treatments, H2L5 hIgG4PE (0.04 or 0.4 mg/kg) andipilimumab or IgG1 (3 mg/kg) or H2L5 hIgG4PE (0.04 or 0.4 mg/kg) andpembrolizumab or IgG4 (5 mg/kg) were dosed. H2L5 hIgG4PE and ipilimumabas well as the matched isotype controls were dosed twice weekly for 6doses, pembrolizumab and isotype control were dosed every 5 days untilend of the H2L5 hIgG4PE dose. For the pharmacodynamic tissue collectioncohorts, H2L5 hIgG4PE was dosed at 0.004, 0.04, 0.4 and 1.2 mg/kg.Treatment groups were evaluated relative to the vehicle and isotypecontrol groups. Treatment groups for vehicle, isotypes and H2L5 hIgG4PEalone and in combination with ipilimumab and pembrolizumab using humanPBMC from donor number #6711 are shown in Table 12. Analysis concludedon day 59 at termination of the study.

TABLE 12 Treatment groups of mice in A2058 melanoma tumor model # mice/Group Treatment 1 Treatment 2 group Dosing 1 Tumor + Vehicle 10 Twice aweek for 6 doses huPBMC (donor #6711) 2 Tumor + Isotype control (IgG1 10IgG1 Twice a week for 6 doses huPBMC 3 mg/kg + IgG4 5 mg/kg) IgG4 every5 days until the end of (donor ICOS dose #6711) 3 Tumor + Ipilimumab 3mg/kg + 10 Twice a week for 6 doses huPBMC IgG4 5 mg/kg IgG4 every 5days until the end of (donor ICOS dose #6711) 4 Tumor + Pembrolizumab 5mg/kg + 10 IgG1 Twice a week for 6 doses huPBMC IgG1 3 mg/kgPembrolizumab every 5 days until (donor the end of ICOS dose #6711) 5Tumor + H2L5 hIgG4PE 10 IgG1 and ICOS Twice a week for huPBMC 0.04mg/kg + IgG1 6 doses (donor 3 mg/kg #6711) 6 Tumor + H2L5 hIgG4PE 0.4mg/kg + 10 IgG1 and ICOS Twice a week for huPBMC IgG1 3 mg/kg 6 doses(donor #6711) 7 Tumor + Ipilimumab 3 mg/kg + 10 Ipilimumab Twice a weekfor 6 huPBMC Pembrolizumab 5 mg/kg doses Pembrolizumab every 5 (donordays until the end of ICOS dose #6711) 8 Tumor + H2L5 hIgG4PE 0.04mg/kg + 10 Ipilimumab and ICOS Twice a huPBMC Ipilimumab 3 mg/kg weekfor 6 doses (donor #6711) 9 Tumor + H2L5 hIgG4PE 0.4 mg/kg + 10Ipilimumab and ICOS Twice a huPBMC Ipilimumab week for 6 doses (donor 3mg/kg #6711) 10 Tumor + H2L5 hIgG4PE 0.04 mg/kg + 10 ICOS Twice a weekfor 6 doses huPBMC Pembrolizumab Pembrolizumab every 5 days until (donor5 mg/kg the end of ICOS dose #6711) 11 Tumor + H2L5 hIgG4PE 0.4 mg/kg +10 ICOS Twice a week for 6 doses huPBMC Pembrolizumab Pembrolizumabevery 5 days until (donor 5 mg/kg the end of ICOS dose #6711) 12 Tumor +IgG4 5 mg/kg 10 Twice a week for 6 doses huPBMC (donor #6711) 13 Tumor +Pembrolizumab 10 Pembrolizumab every 5 days until huPBMC 2.5 mg/kg theend of ICOS dose (donor #6711) 14 Tumor + Pembrolizumab 5 mg/kg 10Pembrolizumab every 5 days huPBMC until the end of ICOS dose (donor#6711) 15 Tumor + H2L5 hIgG4PE 0.4 mg/kg 10 ICOS Twice a week for 6doses huPBMC (donor #6711) 16 Tumor + H2L5 hIgG4PE 0.4 mg/kg + 10 ICOSTwice a week for 6 doses huPBMC Pembrolizumab Pembrolizumab every 5 daysuntil (donor 5 mg/kg + Ipi the end of ICOS dose #6711) 17 Tumor +Vehicle 10 Twice a week for huPBMC pharmacodynamic activity, 5 mice(donor harvested 24 hr post 2nd dose and #6711) 5 mice harvested 24 hrpost 4nd dose 18 Tumor + Isotype Control (IgG4) 10 Twice a week forhuPBMC 1.2 mg/kg pharmacodynamic activity, 5 mice (donor harvested 24 hrpost 2nd dose and #6711) 5 mice harvested 24 hr post 4nd dose 19 Tumor +H2L5 hIgG4PE 0.004 mg/kg 10 Twice a week for huPBMC pharmacodynamicactivity, 5 mice (donor harvested 24 hr post 2nd dose and #6711) 5 miceharvested 24 hr post 4nd dose 20 Tumor + H2L5 hIgG4PE 0.04 mg/kg 10Twice a week for huPBMC pharmacodynamic activity, 5 mice (donorharvested 24 hr post 2nd dose and #6711) 5 mice harvested 24 hr post 4nddose 21 Tumor + H2L5 hIgG4PE 0.4 mg/kg 10 Twice a week for huPBMCpharmacodynamic activity, 5 mice (donor harvested 24 hr post 2nd doseand #6711) 5 mice harvested 24 hr post 4nd dose 22 Tumor + H2L5 hIgG4PE1.2 mg/kg 10 Twice a week for huPBMC pharmacodynamic activity, 5 mice(donor harvested 24 hr post 2nd dose and #6711) 5 mice harvested 24 hrpost 4nd doseEfficacy Study Evaluating H2L5 hIgG4PE Dosed in Combination withIpilimumab or Pembrolizumab

This study was designed to evaluate the anti-tumor efficacy of H2L5hIgG4PE (dosed at 0.01 and 0.04 mg/kg) in combination with ipilimumab orpembrolizumab with matched isotype controls in the human PBMC engraftedNSG mouse using A2058 melanoma tumor model. A total of 13 groups with 10mice per group were assigned into the study. Group 2 was the combinedisotype control of humanized IgG1 and IgG4. H2L5 hIgG4PE was dosed at0.01 mg/kg (Group12) and 0.04 mg/kg (Group13) as single agent. Forcombination treatments, H2L5 hIgG4PE (0.01 and 0.04 mg/kg) andipilimumab or IgG1 (3 mg/kg) or H2L5 hIgG4PE (0.01 and 0.04 mg/kg) andpembrolizumab or IgG4 (5 mg/kg) was dosed. H2L5 hIgG4PE and ipilimumabas well as the matched isotype controls were dosed twice weekly for 6doses, pembrolizumab and isotype control was dosed every 5 days untilend of the H2L5 hIgG4PE dose. A summary of treatment groups, using humanPBMC from donor #4568, is presented in Table 13. Treatment groups wereevaluated relative to the vehicle and isotype control groups.Survivability analysis was concluded on day 33 at termination of thestudy.

TABLE 13 Treatment groups of mice in A2058 melanoma tumor model # mice/Group Treatment1 Treatment2 group Dosing 1 Tumor + Vehicle 10 Twice aweek for 6 doses huPBMC (donor #4568) 2 Tumor + Isotype control (IgG1 10IgG1 Twice a week for 6 doses huPBMC 3 mg/kg + IgG4 5 mg/kg) IgG4 every5 days until the end (donor of ICOS dose #4568) 3 Tumor + Ipilimumab 3mg/kg + 10 Twice a week for 6 doses IgG4 huPBMC IgG4 5 mg/kg every 5days until the end of (donor ICOS dose #4568) 4 Tumor + Pembrolizumab 5mg/kg + 10 IgG1 Twice a week for 6 doses huPBMC IgG1 3 mg/kgPembrolizumab every 5 days (donor until the end of ICOS dose #4568) 5Tumor + H2L5 hIgG4PE 0.01 mg/kg + 10 IgG1 and ICOS Twice a week huPBMCIgG1 3 mg/kg for 6 doses (donor #4568) 6 Tumor + H2L5 hIgG4PE 0.04mg/kg + 10 IgG1 and ICOS Twice a week huPBMC IgG1 3 mg/kg for 6 doses(donor #4568) 7 Tumor + Ipilimumab 3 mg/kg + 10 Ipilimumab Twice a weekfor 6 huPBMC Pembrolizumab doses Pembrolizumab every 5 (donor 5 mg/kgdays until the end of ICOS dose #4568) 8 Tumor + H2L5 hIgG4PE 10Ipilimumab and ICOS Twice a huPBMC 0.01 mg/kg + week for 6 doses (donorIpilimumab 3 mg/kg #4568) 9 Tumor + H2L5 hIgG4PE 0.04 mg/kg + 10Ipilimumab and ICOS Twice a huPBMC Ipilimumab week for 6 doses (donor 3mg/kg #4568) 10 Tumor + H2L5 hIgG4PE 0.01 mg/kg + 10 ICOS Twice a weekfor 6 doses huPBMC Pembrolizumab Pembrolizumab every 5 days (donor 5mg/kg until the end of ICOS dose #4568) 11 Tumor + H2L5 hIgG4PE 0.04mg/kg + 10 ICOS Twice a week for 6 doses huPBMC PembrolizumabPembrolizumab every 5 days (donor 5 mg/kg until the end of ICOS dose#4568) 12 Tumor + H2L5 hIgG4PE 10 Twice a week for 6 doses huPBMC 0.01mg/kg (donor #4568) 13 Tumor + H2L5 hIgG4PE 0.04 mg/kg 10 Twice a weekfor 6 doses huPBMC (donor #4568)

Statistical Analysis

The event for survival analysis was tumor volume >2000 mm³, tumorulceration, mouse body weight loss>20%, moribund or found dead,whichever came first. The exact time to cut-off volume was estimated byfitting a linear line between log tumor volume and day of twoobservations, the first observation that exceed the cut-off volume andthe one observation that immediately preceded the cut-off volume.Kaplan-Meier (KM) method was carried out to estimate the survivalprobability of different treatment groups at a given time. The mediantime to endpoint and its corresponding 95% confidence interval wasreported. Whether or not KM survival curves are statistically differentbetween any two groups was then tested by log-rank test.

Tumor volume data from the last day in which there were 10 animals pergroup (i.e. before any animals were euthanized) was utilized to maketumor volume comparisons between the different treatment groups. Priorto the analysis, the tumor volume was natural log transformed due to theinequality of variance in the different treatment groups. ANOVA followedby pair-wise comparison were then carried out on the log transformeddata.

Graphpad Prism software was used to plot the tumor growth and bodyweight data.

Results

H2L5 hIgG4PE Dose Response (FIG. 20A)

Tumor Growth Inhibition:

Control group: Human PBMC (donor 7129) showed no effect on A2058 tumorgrowth in NSG mice. A2058 tumor bearing mice with or without human PBMC,A2058 tumor bearing mice with human PBMC treated with vehicle andisotype control antibodies developed tumors that progressed as expected(Group#1 vs. Group#10, Group#1 vs. Group#2, Group#1 vs. Group#4, p=1).Ipilimumab treatment at 3 mg/kg (Group #3) demonstrated significanttumor growth inhibition (p<0.03) as compared to vehicle control Group#1,however the statistical significance was lost (p<0.22) when compared tothe isotype control Group #2. This indicated the isotype antibody mayaffect tumor growth.H2L5 hIgG4PE treatment at 0.4 mg/kg demonstrated a trend of tumor growthinhibition and increased survivability of mice compared to other doses,although the affects were not statistically significant when compared toeither vehicle or isotype control.

Clinical Observations:

Loss of body weight in mice was observed during the study which wasapproximately 20% at the end of study. It has been reported that bothGvHD and tumor burden can result in a drop in mice body weight, thoughin this study the body weight loss seemed to be more related to A2058tumor since tumor bearing mice without PBMC engraftment (group #10)showed the same trend. Tumor ulceration was observed in multiple tumorsduring the study, including the isotype control group.

Mouse Fates:

Most mice were removed upon tumors reaching volumes >2000 mm³. Threemice were euthanized due to tumor ulceration, and three mice wereeuthanized due to body weight loss of >20%. Nine mice were found deadrandomly across the groups, including two in the vehicle, and threetotal in the isotype control groups. These deaths were attributed to thesusceptibility of the model for a Graft-versus-Host Disease state, andnot treatment related since no pattern was observed with treatmentgroups compared to vehicle or isotype control groups.Efficacy study with H2L5 hIgG4PE in combination with ipilimumab andPembrolizumab (FIG. 20B)

Tumor Growth Inhibition:

Control group: A2058 tumor bearing mice with human PBMC treated withvehicle or isotype control antibodies developed tumors which grew asexpected.

Monotherapy:

Ipilimumab treatment at 3 mg/kg combined with IgG4 (Group#3) resulted insignificant tumor growth inhibition (p<0.04) as compared to vehiclecontrol Group#1. However, when compared to isotype control Group#2, thestatistical significance was lost (p<0.23).Pembrolizumab treatment alone at 2.5 or 5 mg/kg (Group#13, 14) showedobservable tumor growth inhibition without statistical significance whencompared to vehicle or isotype control group#12. Pembrolizumab combinedwith IgG1 (Group#4), showed observable tumor growth inhibition withoutstatistical significance, however a significant increase in survival wasobserved (p<0.04) as compared to vehicle control Group#1. Statisticalsignificance was lost (p<0.4) when compared with isotype controlGroup#2.H2L5 hIgG4PE treatment alone at 0.4 mg/kg (Group#15) showed observabletumor growth inhibition without statistical significance as compared tovehicle or isotype control group#12. H2L5 hIgG4PE at 0.04 or 0.4 mg/kgcombined with IgG1 (Group#5 and 6) showed observable delay in tumorprogression and mice survival but didn't reach statistical significance.

Combination Treatment:

Combination of H2L5 hIgG4PE (0.04 or 0.4 mg/kg) with ipilimumab (3mg/kg). Groups #8 and #9 showed no additional tumor growth inhibition ascompared to Ipilimumab alone (Group#3). Combination of H2L5 hIgG4PE(0.04 or 0.4 mg/kg) with pembrolizumab (5 mg/kg) Groups #10 and #11demonstrated modest but insignificant tumor growth inhibition and micesurvival compared to pembrolizumab monotherapy, Group#4, or H2L5 hIgG4PEmonotherapy Groups #5 and #6.

Clinical Observations:

Mice body weight loss observed during the study was approximately 20%.Tumor ulceration was apparent in multiple tumors during the study acrossthe majority of group.

Mouse Fates:

A total of 100 out of 160 mice were euthanized when tumor volumesreached >2000 mm³. 29 mice were euthanized due to tumor ulceration, 18mice were found dead, 12 mice were euthanized due to body weightloss >20%, and one mouse was euthanized as moribund. Mice were founddead across the groups including the isotype control group #2. Thesedeaths were attributed to the susceptibility of the model for aGraft-versus-Host Disease state, and not treatment related since nopattern was observed with treatment groups compared to the isotypecontrol group.Efficacy study evaluating H2L5 hIgG4PE dosed in combination withipilimumab or pembrolizumab (FIG. 20C)

Tumor Growth Delay:

Control group: A2058 tumor bearing mice with human PBMC treated withvehicle or isotype control antibodies developed tumors which grew asexpected.

Monotherapy:

Ipilimumab treatment at 3 mg/kg combined with IgG4 (Group#3)demonstrated significant tumor growth inhibition (p<0.02) andsignificant increase in survival (p<0.01) as compared to vehicle controlGroup#1. Compared to isotype control Group#2 however, the tumor growthinhibition did not reach significance (p<0.13) while significantincrease in mice survival remained (p<0.04).Pembrolizumab treatment at 5 mg/kg combined with IgG1 (Group#4) showedtumor growth inhibition without statistical significance as compared tovehicle or isotype control Group#2.H2L5 hIgG4PE treatment alone at 0.01 mg/kg or 0.04 mg/kg (Group#12 and#13) demonstrated significant tumor growth inhibition (p<0.03) comparedto vehicle control group #1 H2L5 hIgG4PE dosed at 0.04 mg/kg also showeda significant increase in mice survival (p<0.048) as compared to vehiclecontrol group#1. However, as compared to isotype control group#2, tumorgrowth inhibition and survival did not reach statistical significancefor groups #12 and #13. H2L5 hIgG4PE at 0.01 mg/kg combined with IgG1(Group#5) showed significant tumor growth inhibition (p<0.03) and micesurvival (p<0.03) as compared to vehicle control group#1. However, ascompared to isotype control group#2, tumor growth delay and survival didnot reach statistical significance. H2L5 hIgG4PE at 0.04 mg/kg combinedwith IgG1 (Group#6) showed observable tumor growth inhibition and micesurvival, but did not reach statistical significance.

Combination Treatment:

The combination of H2L5 hIgG4PE with ipilimumab (0.01 mg/kg plusipilimumab 3 mg/kg; Group#8) showed observable tumor growth inhibitionand mice survival but failed to reach statistical significance. H2L5hIgG4PE combination with ipilimumab (0.04 mg/kg plus ipilimumab 3 mg/kg;Group#9) demonstrated significant tumor growth inhibition (p<0.00) and asignificant increase in mice survival (p<0.04) as compared to vehiclecontrol group#1 or isotype control group#2 (p<0.02). However, ascompared to isotype control survival failed to reach statisticalsignificance. Combination activity did not reach significance ascompared to monotherapy ipilimumab group#3 or H2L5 hIgG4PE monotherapygroups.

H2L5 hIgG4PE (0.01 mg/kg or 0.04 mg/kg) combination with pembrolizumab(5 mg/kg), Groups#10 and #11, showed significant tumor growthinhibition. (p<0.03) and significant increase of mice survival observed(p<0.03) when comparing to vehicle control group#1. When comparing toisotype control group#2, the tumor growth inhibition significanceremained in the 0.04 mg/kg H2L5 hIgG4PE combination with pembrolizumab(p<0.03). The survival benefit failed to reach statistical significancehowever. The combination failed to reach significance as compared toeither monotherapy treatment group pembrolizumab group#3 or H2L5 hIgG4PEgroup#5 or #6. Thus, H2L5 hIgG4PE combined with pembrolizumab (0.01 or0.04 mg/kg plus pembrolizumab 5 mg/kg) demonstrated an increase in tumorgrowth inhibition and mice survival but failed to reach statisticalsignificance versus isotype control or monotherapies.

Clinical Observations:

Mice body weight loss observed during the study was approximately 20%.Tumor ulceration was observed across the majority of groups during thestudy.

Mouse Fates:

A total of 91 mice were euthanized due to tumor size >2000 mm³, 34 micewere euthanized due to tumor ulcerations, and 5 mice were found dead.These deaths were attributed to the susceptibility of the model for aGraft-versus-Host Disease state.

Discussion

Efficacy of H2L5 hIgG4PE as a monotherapy and in combination withpembrolizumab as well as ipilimumab was evaluated in the human PBMCengrafted NSG mouse model with A2058 melanoma tumors. This model wherehuman PBMC are intravenously injected into adult immunodeficient NSG(NOD/SCID/IL-2Rγnull) mice is known as the Hu-PBMC NSG model. It inducesa Graft-versus-Host Disease (GvHD) and has been used to study effectorand memory T cell activity. The Hu-PBMC NSG model was implanted withhuman cancer cell line A2058 subcutaneously to investigate the effect ofhuman immunotherapeutic antibodies on tumor growth. The limitations ofthis model include onset of GvHD symptoms, loss of body weight, andfrequent tumor ulcerations which prevent survival monitoring for longerperiod of time as is possible with syngeneic mouse tumor models.

Initial studies evaluating H2L5 hIgG4PE at doses ranging from 0.04 mg/kgto 4 mg/kg showed that doses in the lower range demonstrated modesttumor growth inhibition. Delay in tumor progression and increasedsurvival of mice was observed in dose groups ranging from 0.04 to 0.4mg/kg though not statistically significant when compared to the isotypecontrol groups. Based on these studies, H2L5 hIgG4PE doses of 0.04 to0.4 mg/kg were selected for further evaluation alone and in combinationwith pembrolizumab and ipilimumab in two studies with PBMC grafts fromtwo different donors (donor numbers 4568 and 6711). Modest responses forH2L5 hIgG4PE monotherapy and combination with pembrolizumab wereobserved in one of the two combination studies performed. Thecombination study using PBMC donor 4568 (Table 13, FIG. 20C)demonstrated anti-tumor activity of the monotherapy and combinationwhile the study using PBMC donor 6711 (Table 12, FIG. 20B) did not showsignificant anti-tumor effect, which likely was a result of donor PBMCdifferences between studies, which reflect the patient responsevariability that may be observed in the clinic. In this secondcombination study with PBMC donor 4568, enhanced tumor growth inhibitionand increased survivability of mice was observed in the combinationgroup when compared to either agent alone, although this difference wasnot statistically significant. Combination synergy was observed however,since the H2L5 hIgG4PE 0.04 mg/kg dose in combination with pembrolizumab5 mg/kg resulted in a statistically significant decrease in tumor volumeten days post first dose and increased survivability versus the isotypecontrol group (p<0.05), while the monotherapies did not. In fact, 50% ofthe mice in the H2L5 hIgG4PE and pembrolizumab combination groupremained on study by day 33, but were removed due to tumor ulcerations.Only four mice were removed from study due to tumor volume from thiscombination group, while 8 to 9 mice were removed from study in thepembrolizumab and isotype groups.

Anti-PD1 therapy did not demonstrate statistically significant activityin this model as seen with the limited change in tumor growth andsurvival seen with pembrolizumab treated cohort compared to isotopetreated cohort. Ipilimumab monotherapy showed a trend of tumor growthinhibition modestly better than pembrolizumab in both studies, and itshowed statistically significant increase in survival versus isotype inthe second combination study with the responsive PBMC donor 4568(p<0.04). The H2L5 hIgG4PE 0.01 mg/kg dose in combination withipilimumab 3 mg/kg showed a significant increase in survival versusipilimumab (p<0.02), but not versus H2L5 hIgG4PE monotherapy. There wereno additional significant effects on tumor volume observed with thecombination of H2L5 hIgG4PE and ipilimumab in this model compared toeither agent alone. Mice from across all treatment groups includingvehicle and isotype control groups were found dead as reported in theFate Tables. These deaths were attributed to the susceptibility of themodel for a Graft-versus-Host Disease state, and not treatment related.

Example 12 Functional Activity of Anti-Murine ICOS Agonist AntibodyAlone and in Combination with Anti-PD1 and Anti-CTLA-4 Antibodies InVivo CT26 and EMT6 Syngeneic Mouse Tumor Models CT26 Murine ColonCarcinoma Mouse Tumor Model Methods

This study was conducted under a protocol which was approved by the GSKInstitutional Animal Care and Use Committee prior to commencement of thestudy.

Animals

In this study 164 female BALB/c mice from Harlan Sprague Dawley. Micewere 6-8 weeks old at the beginning of the study when they wereinoculated.

Cell Culture and Inoculation

One vial of CT-26 cells (ATCC: CRL-2638) (3×10⁶ cells; P-11) was thawedfrom −140° C. and plated in RPMI with 10% FBS. Cells were subcultured 3times over 10 days. Trypsin/EDTA was used to facilitate cell detachmentfrom culture flask during subculturing. Cells were collected, washedtwice, and re-suspended in RPMI without FBS at 5×10⁵ cells/ml. Mice wereinoculated subcutaneously with 0.1 ml cells (5×10⁴ cells/mouse) on theright hind flank.On the day of cell collection and inoculation, cell counts were done onBeckman Coulter Vi-cell XR and checked by hemacytometer. Cells weredetached from flask with trypsin/EDTA and washed twice, first withRPMI+10% FBS and second with RPMI only and resuspended in 10 ml RPMI.178×10⁶ cells were collected in 20 ml RPMI with 98.8% viability. 1.685ml cell suspension (15×10⁶ cells total) was added to 28.315 ml RPMI.15×10⁶ cells/30 ml media=5×10⁵ cells/ml. This equates to 5×10⁴ cells/100μl.

Antibody Formulation and Preparation

Antibodies were diluted from stock source vials to desiredconcentrations in sterile 0.9% saline on the day of dosing. Anti-ICOSagonist clone C398.4 was tested at 0.05 mg/kg and 0.5 mg/kg. Each dosewas also tested with both anti-PD1 10 mg/kg and anti-CTLA-4 1 mg/kg.

Experimental Protocol(s) Tumor Monitoring and Dosing

Mice were inoculated on day 0. On day 11 body weight and tumor volumewere measured. Mice were randomized into the 12 study groups shown inTable 14 with 10 mice/group based on tumor size. Randomization was doneusing Studylog Study Director software. Mice were dosed based on thestudy design chart twice weekly starting on randomization day andcontinuing for 6 total doses. Dosing was interperitoneal (IP) in 100 μlvolume of 0.9% saline vehicle. Tumor volume and body weight weremeasured 3 times per week throughout the study.

Endpoints

Mice were removed from the study for tumor burden when tumor volume wasgreater than 2000 mm³. Tumor volume was calculated by applying lengthand width caliper measurements to the following formula: TV=0.52*L*W².

Additionally mice were removed from study when tumors developed openulcerations. Ulcerations were observed throughout the experiment,however scabbed over ulcerations alone were not an endpoint unless theyformed open holes.

Although it did not apply to any mice in this study a third endpointestablished at the beginning of the study was a decrease of 20% bodyweight.

Drugs and Materials

Antibody Vendor Catalog # Lot Clone ICOS Biolegend 93108 B205973 C398.4PD1 BioXcell BE0146 5792-10/0815B RMP1-14 CTLA-4 BioXcell BE01645632-4/0715 9D9 Mouse IgG2b BioXcell BE0086 4700/1014 MCP-11 Rat IgG2aBioXcell BE0089 5679-6/0815 2A3 Hamster IgG Biolegend 92257 B205974HTK888All antibodies were diluted to desired concentrations in 0.9% saline andsaline was used as a vehicle control.

Data Analysis

The event for survival analysis is tumor volume of 2000 mm³ or tumorulceration, whichever came first. The exact time to cut-off volume wasestimated by fitting a linear line between log tumor volume and day oftwo observations, the first observation that exceed the cut-off volumeand the one observation that immediately preceded the cut-off volume.The Kaplan-Meier (KM) method was carried out to estimate the survivalprobability of different treatment groups at a given time. The mediantime to endpoint and its corresponding 95% confidence interval wasreported. Whether or not KM survival curves are statistically differentbetween any two groups was then tested by the log-rank test.

Tumor volumes at 17 days after initial dosing between the differenttreatment groups were compared. Prior to the analysis, the tumor volumewas natural log transformed due to the inequality of variance in thedifferent treatment groups. ANOVA followed by pair-wise comparison werethen carried out on the log transformed data.

TABLE 14 Study Groups Group No. Treatment 1 Saline 2 Mouse IgG2b 20 μg +Hamster IgG 10 μg 3 Rat IgG2a 200 μg + Hamster IgG 10 μg 4 Hamster IgG10 μg 5 ICOS 1 μg 6 ICOS 10 μg 7 CTLA-4 20 μg 8 PD1 200 μg 9 ICOS 1 μg +CTLA-4 20 μg 10 ICOS 10 μg + CTLA-4 20 μg 11 ICOS 1 μg + PD1 200 μg 12ICOS 10 μg + PD1 200 μg

The raw p-value, as well as the false discovery rate (FDR) adjustedp-values, from the comparisons of days to events by survival analysisand the comparisons of log transformed tumor volume at day 10 betweentreatment groups are shown in the above table. Comparisons, using FDRadjusted p-values<0.05, are declared to be statistically significant.

Results

Mouse fate tracking showed that the number of mice removed from studyfor tumor burden and tumor ulceration. All remaining mice are tumor freeat study day 61 except 1 mouse in G7 which has a tumor volume of 579.16mm³.

For survival (time to endpoints) groups 9 and 12 showed significantincrease in survival compared to the vehicle control group (p=0.008 andp=0.001 respectively). Group 12 showed statistically significantextended survival compared to groups 2, 4, and 5 (p=0.006, 0.001, 0.02).However, no combination group showed statistically significant (p<0.05)increased survival over either monotherapy. (FIG. 21)

Discussion

The combination therapy groups, particularly the high dose anti-ICOS andanti-PD1 combination (Group 12), demonstrated tumor growth inhibitionand increased survival over monotherapy and isotype control groups,although statistical significance was not reached at Day 61. The isotypecontrol for group 12 was the Rat IgG2a+Hamster IgG group 3. Themonotherapy groups for comparison are; ICOS 10 μg (group 6) and PD1 200μg (group 8). A total of 5 mice remained as tumor free in group 12compared to 1 in group 3, 1 in group 6 and 1 in group 8. The survivalbenefit was quantified by taking the day each mouse reached any of thepre-determined study endpoints. A number of mice were removed from studyfor open tumor ulcerations and not due to tumor burden.

In the high dose ICOS+CTLA-4 combination group (group 10) an increasednumber of mice were removed due to tumor ulceration by day 31 whichlikely masked the survival and anti-tumor benefit that this combinationprovided. In this group, 5 mice were removed for tumor ulcerations andonly 2 for tumor burden reaching 2000 mm³. All tumors removed due totumor ulceration where still at modest size when taken off study, and itis expected that tumor ulceration may have been the result of atherapy-induced anti-tumor immune response in these mice. Three miceremained tumor free in this group out to day 61. The 2 mice removed fortumor burden were the lowest number of mice removed for tumor burden ofall groups.

EMT6 Mammary Carcinoma Mouse Tumor Model Experimental Protocol(s)

All procedures and euthanization criteria described in this document arein accordance with IACUC protocol AUP0606. Animals are weighed andinoculated on the right hind quarter with 100 μl of 1×10⁵ EMT6 tumorcells per mouse. The number of mice inoculated is equal to at least 130%of what was needed for the study. Assuming 30% failure rate (either toobig or too small at time of start of study), the goal was to have n=10for each group. After tumor cell inoculation, tumor growth and totalbody weight were measured 3 times a week with a Fowler “ProMax” digitalcaliper for 4 weeks or longer. Antibodies were acquired from acommercial vendor and diluted to desired concentration in 0.9% saline.Dosing (i.p.) occurred biweekly, for a total of 6 doses and initiated onthe day of randomization, designated as Day 0, when average tumor volumeapproximated 100 mm³, approximately 7 to 8 days after inoculation.Randomization was performed using the Studylog Study Director Suitesoftware. Length and width of tumors was measured in order to determinetumor volume using the formula (tumor volume=L*W²*0.52). Tumormeasurement of greater than 2,000 mm³ for an individual animal resultedin removal from study. Mice may also be removed from the study due toweight loss (>20%), tumor ulceration, or any other obvious inhibition ofnormal mouse activity due to morbidity.

In this study, a total of 191 animals were inoculated with EMT6 cells inorder to generate enough mice with tumors in the desired size range for13 groups of 10 mice each as shown in Table 15. Saline vehicle injectedmice and isotype control groups served as controls for ICOS, PD1 andCTLA-4 mAb treated mice. The isotype control for ICOS (Hamster IgG) wasdosed at 10 μg alone and in combination with the isotype for CTLA-4(mouse IgG2b) or PD-1 (rat IgG2a). Monotherapy treatment groups foranti-CTLA-4 (9D9) and anti-PD-1 (RMP1-14) were dosed at 20 and 200 μgper mouse, respectively, and evaluated in combination with the ICOSisotype control. The C398.4 clone of ICOS agonist was dosed at 10 and 1μg per mouse. Efficacy of the ICOS agonist was also evaluated at 10 and1 μg per mouse dosed in combination with anti-CTLA-4 or anti-PD-1. Anadditional group of PD-1 and CTLA-4 at predescribed concentrations wasincluded as a positive control comparator group. Statistical analysis oftumor volume was performed on day 13 post randomization. Survivabilityanalysis included mice on study through day 60.

TABLE 15 Study Groups Dosing treatment 1 treatment 2 n = Group 1: 1 ×10⁵ cells per saline 10 Group 2: 1 × 10⁵ cells per Hamster IgG 10 μgmIgG2b 20 μg 10 Group 3: 1 × 10⁵ cells per Hamster IgG 10 μg rIgG2a 200μg 10 Group 4: 1 × 10⁵ cells per Hamster IgG 10 μg 10 Group 5: 1 × 10⁵cells per ICOS 10 μg 10 Group 6: 1 × 10⁵ cells per ICOS 1 μg 10 Group 7:1 × 10⁵ cells per CTLA4 20 μg Hamster IgG 10 10 μg Group 8: 1 × 10⁵cells per PD-1 200 μg Hamster IgG 10 10 μg Group 9: 1 × 10⁵ cells perICOS 10 μg CTLA4 20 μg 10 Group 10: 1 × 10⁵ cells per ICOS 1 μg CTLA4 20μg 10 Group 11: 1 × 10⁵ cells per ICOS 10 μg PD-1 200 μg 10 Group 12: 1× 10⁵ cells per ICOS 1 μg PD-1 200 μg 10 Group 13: 1 × 10⁵ cells perCTLA4 20 μg PD-1 200 μg 10

Drugs and Materials Animals

Female Balb/c mice from 6 to 8 weeks of age were received from HarlanSprague Dawley and housed in accordance with IACUC standards.

EMT6 Cells

EMT6 cells were thawed and cultured in cell culture flasks for eightdays prior to inoculation. Cells were passed 3 times in this time. Onthe day of inoculation, the cells are harvested from the flask incomplete medium. Cells are centrifuged and resuspended in Weymouth's(with 15% FBS). This step is repeated 3 times in Weymouth's mediawithout FBS. Cell density and viability are checked via trypan blueexclusion. Cells are then diluted to desired density (1×10⁶ cells permL).

Immunotherapeutics

All therapeutics were diluted to desired concentrations in 0.9% sodiumchloride on the day of dosing and injected i.p. using a 30 G needle.Therapeutic and control dilutions are presented below in Table 16.

TABLE 16 Therapeutic dilutions starting desired dose/ number volume addTotal total conc. conc. dilution mouse of needed stock diluent volume Rxmg/mL mg/mL 1: mg mice mL mL mL mL mouse 4.46 0.1 44.6 0.02 10 2 0.104.36 4.46 IgG2b rat 6.92 1 6.92 0.2 10 2 0.40 2.37 2.77 IgG2a Hamster1.47 0.05 29.4 0.01 50 10 0.40 11.36 11.76 IgG CTLA4 6.1 0.1 61 0.02 408 0.15 9 9.15 PD-1 7.44 1 7.44 0.2 40 8 1.30 8.372 9.672 ICOS 5 0.05 1000.01 30 6 0.10 9.9 10 ICOS 0.05 0.005 10 0.001 30 6 1.00 9 10

Data Analysis

Statistical analysis

The event for survival analysis was tumor volume of 2000 mm³ or tumorulceration, whichever came first. The exact time to cut-off volume wasestimated by fitting a linear line between log tumor volume and day oftwo observations, the first observation that exceed the cut-off volumeand the one observation that immediately preceded the cut-off volume.The Kaplan-Meier (KM) method was carried out to estimate the survivalprobability of different treatment groups at a given time. The mediantime to endpoint and its corresponding 95% confidence interval wasreported. Whether or not KM survival curves were statistically differentbetween any two groups was then tested by the log-rank test.

Tumor volumes at 13 days after initial dosing between the differenttreatment groups were compared. Prior to the analysis, the tumor volumewas natural log transformed due to the inequality of variance in thedifferent treatment groups. ANOVA followed by pair-wise comparison werethen carried out on the log transformed data. SAS 9.3 and R 3.0.2Analysis Software was utilized.

Results

Balb/c mice were inoculated and randomized into groups of ten based ontreatment regimen 8 days later. Administration of therapeutics orcontrols began on randomization day (Day 0) and continued twice a weekfor 3 weeks.

The saline treated group grew tumors at the expected rate relative toprevious EMT-6 studies. All mice in the saline vehicle group wereeuthanized due to tumor size or ulceration by day 30. Treatment withhamster IgG alone or in combination with rat IgG2a or mouse IgG2b,resulted in no statistically significant change in average tumor growthor survival when compared to the saline vehicle group.

At 13 days post randomization, the ICOS monotherapy groups demonstratedlittle observable change in average tumor growth as compared to isotypecontrols. However, the high dose ICOS treatment group (10 μg)demonstrated an apparent trend towards more tumor growth inhibition thanthe low dose group. An effect that was comparable to the CTLA-4monotherapy activity was observed. Monotherapy treatment with PD-1 mAbalso resulted in some observable, but statistically insignificantreduction in average tumor volume at day 13. However, as with ICOS andCTLA-4 monotherapy, this did not result in increased survival whencompared to that of the appropriate isotype groups. Treatment with thecombination of anti-PD-1 and anti-ICOS antibody clone C398.4 at the 10μg dose resulted in considerable tumor growth inhibition as compared tocontrol and monotherapy treatment groups (FIG. 22). Three mice in thiscombination group achieved complete tumor regression, a considerableimprovement over control or monotherapy treatment groups. However, dueto the statistical criteria used, statistically significant improvementin survival was not reached. The combination of anti-PD-1 with 1 μg ofICOS agonist antibody clone C398.4 did result in a statisticallysignificant decrease in average tumor growth at day 13 as compared tosaline vehicle control (p<0.05) and ICOS monotherapy (p<0.05) groups of1 and 10 μg. Four mice from this treatment regimen achieved completetumor regression resulting in significant trend towards increasedsurvival that failed to reach statistical significance.

The ICOS antibody at both doses in combination with anti-CTLA-4demonstrated little observable benefit in tumor growth inhibition orsurvival as compared to monotherapy treatment with either antibody.

Discussion

While isotype controls resulted in no obvious change in average tumorvolume or overall survival when compared to the saline vehicle group,there were individual animals in the hamster IgG group (group 4) and thehamster IgG and rat IgG2a (group 3) that demonstrated delayed tumorgrowth. In the hamster IgG & rat IgG2a isotype group, one mouse survivedbeyond the last saline vehicle mouse, being sacrificed on day 36 due toulceration with a tumor that measured 1156.56 mm³ in volume. Two mice inthe hamster IgG group survived longer than the saline group. One animalwas euthanized due to tumor size on day 36, and the second one on day 41due to ulceration with a measurement of 1899.28 mm.

The dosing regimen of anti-PD-1 with 10 μg of anti-ICOS agonist led toan observable inhibition of tumor growth resulting in a decrease intumor volume at day 13 when compared to isotype controls, although thisdifference was less obvious when compared to anti-PD-1 monotherapy.However, the combination did result in a total of five animals survivingbeyond any in the anti-PD-1 monotherapy group, with three miceexperiencing complete tumor regression as compared to none in theanti-PD-1 monotherapy group.

Pairing anti-PD-1 with a 1 μg dose of ICOS agonist antibody led to anobservable decrease in average tumor size at day 13 when compared toisotype controls and respective monotherapy groups. This decrease wasstatistically significant when compared to saline vehicle control(p<0.05) and the 1 μg ICOS monotherapy group (p<0.05). Four miceexperienced complete tumor regression and survived beyond any in thePD-1 monotherapy group

The survival benefit observed with the ICOS+PD1 combination group wasnot found to reach statistical significance relative to controls by day60. However, the tumor growth inhibition and survival benefit of theICOS+PD1 combination treatment groups was comparable to the activityobserved with the PD1+CTLA-4 combination group, which was considered apositive control for anti-tumor activity in this study. This suggeststhat a combination of ICOS and PD1 antibodies may have benefit similarto CTLA-4 and PD1 combinations, which have demonstrated significantclinical activity in some tumor types.

Of the 130 mice enrolled in this study, 12 remained alive at day 60 with11 having achieved complete tumor regression. Of the 118 mice that metendpoints for study removal, 111 were removed due to reaching a tumorsize of 2000 mm³. The remaining seven mice were euthanized due toulceration on the tumor. Occurrences of ulceration were spread out amongthe groups. Groups 1 (Saline), 3 (hamster IgG & rat IgG2a), 4 (hamsterIgG), 6 (1 μg ICOS), and 10 (CTLA-4 with 1 μg ICOS) all had one animalremoved due to ulceration. Group 13 (CTLA-4+PD-1) showed two animalssacrificed due to ulceration. The remaining groups had no animalsremoved due to ulceration.

1. An ICOS binding protein or antigen binding portion thereof comprisinga V_(H) domain comprising an amino acid sequence at least 90% identicalto the amino acid sequence set forth in SEQ ID NO:7; and a V_(L) domaincomprising an amino acid sequence at least 90% identical to the aminoacid sequence as set forth in SEQ ID NO:8 wherein said ICOS bindingprotein or antigen binding portion thereof specifically binds to humanICOS.
 2. The ICOS binding protein or antigen binding portion thereof ofclaim 1 comprising heavy chain CDRs having the amino acid sequences setforth in SEQ ID NO:1; SEQ ID NO:2; and SEQ ID NO:3 and light chain CDRshaving the amino acid sequences set forth in SEQ ID NO:4; SEQ ID NO:5;and SEQ ID NO:6.
 3. The ICOS binding protein or antigen binding portionthereof of claim 1 comprising a V_(H) domain comprising an amino acidsequence set forth in SEQ ID NO:7; and a V_(L) domain comprising theamino acid sequence set forth in SEQ ID NO:8.
 4. The ICOS bindingprotein or antigen binding portion thereof of claim 1 that is an agonistto human ICOS.
 5. The ICOS binding protein or antigen binding portionthereof of claim 1 further comprising an IgG4 isotype scaffold or avariant thereof.
 6. The ICOS binding protein or antigen binding portionthereof of claim 1 further comprising a hIgG4PE scaffold.
 7. The ICOSbinding protein or antigen binding portion thereof of claim 1 whereinsaid ICOS binding protein is a humanized monoclonal antibody.
 8. Apharmaceutical composition comprising the ICOS binding protein orantigen binding portion thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 9. A polynucleotide encoding the ICOS bindingprotein or antigen binding portion thereof of claim
 1. 10. An isolatedhumanized monoclonal antibody comprising a V_(H) domain comprising anamino acid sequence set forth in SEQ ID NO:7; a V_(L) domain comprisingan amino acid sequence set forth in SEQ ID NO:8; and a hIgG4 scaffold ora variant thereof.
 11. The isolated humanized monoclonal antibody ofclaim 10 wherein the hIgG4 scaffold is hIgG4PE.
 12. An ICOS bindingprotein or antigen binding portion thereof, wherein the ICOS bindingprotein or antigen binding portion thereof cross-competes for bindingfor human ICOS with a reference antibody or antigen binding portionthereof comprising a V_(H) domain comprising an amino acid sequence setforth in SEQ ID NO:7; and a V_(L) domain comprising the amino acidsequence set forth in SEQ ID NO:8.